Functionalized Carbon Nanotubes for Biomedical Applications 1119904838, 9781119904830

Table of contents :
Cover
Title Page
Copyright Page
Contents
Preface
Part 1: Overview of Functionalized Carbon Nanotubes
Chapter 1 Functionalized Carbon Nanotubes: An Introduction
1.1 Introduction
1.2 Carbon Nanotube’s Classification
1.3 Structural and Morphological Analysis of Carbon Nanotubes
1.4 Synthetic Techniques of Carbon Nanotubes
1.5 Functionalization of Carbon Nanotubes
1.6 Commercial Scale Use of Functionalized Carbon Nanotubes
1.7 Conclusion and Future Prospects
References
Chapter 2 Functionalized Carbon Nanotubes: Synthesis and Characterization
2.1 Introduction
2.2 Synthesis Methods
2.2.1 Arc Discharge
2.2.2 Laser Ablation
2.2.3 Chemical Vapor Deposition
2.3 Characterization
2.3.1 Raman Spectroscopy
2.3.2 Fourier Transform Infrared Spectroscopy (FT-IR)
2.3.3 Thermogravimetric Analysis (TGA)
2.3.4 Scanning Electron Microscopy (SEM)
2.3.5 Transmission Electron Microscopy (TEM)
2.3.6 X-Ray Diffraction (XRD)
2.3.7 X-Ray Photoelectron Spectroscopy (XPS)
2.4 Functionalized Routes of CNTs
2.4.1 Surface Oxidation
2.4.2 Doping Heteroatoms
2.4.3 Alkali Activation
2.4.4 Sulfonation
2.4.5 Halogenation
2.4.6 Grafting
2.4.6.1 Grafting via Oxygen-Containing Groups
2.4.6.2 Grafting via Diazonium Compounds
2.4.6.3 Other Grafting Methods
2.4.7 Non-Covalent Functionalization of CNTs
2.4.8 Deposition on Functionalized CNTs
2.4.9 Physiochemical Approaches
2.4.10 Electrochemical Deposition
2.4.11 Electroless Deposition
2.5 Conclusion
References
Chapter 3 Carbon Nanotubes: Types of Functionalization
3.1 Introduction
3.2 Carbon Nanotubes
3.3 Functionalization of Carbon Nanotubes
3.3.1 Covalent Functionalization
3.3.2 Non-Covalent Functionalization of Carbon Nanotubes
3.3.2.1 Reversibility in Non-Covalent Functionalization
3.3.2.2 Solvent Variation in Non-Covalent Functionalization
3.3.3.3 pH of the System in Non-Covalent Functionalization
3.3.3.4 Temperature Responsive System in Non-Covalent Functionalization
3.4 Conclusion and Future Outlook
Acknowledgements
Web Links
References
Chapter 4 Functionalization Carbon Nanotubes Innovate on Medical Technology
4.1 Introduction
4.2 Functionalization CNTs for Biomedical Applications
4.3 Potential Applications of CNTs in Cancer Therapy
4.3.1 Anti-Tumor Immunotherapy
4.3.2 Anti-Tumor Hyperthermia Therapy
4.3.3 Anti-Tumor Chemotherapy
4.3.4 Other Cancer Treatment Strategies
4.4 Treatment of Central Nervous System Disorders
4.5 Treatment of Infectious Diseases
4.6 CNTs-Based Transdermal Drug Delivery
4.7 f-CNTs for Vaccination
4.8 Application of f-CNTs in Tissue Engineering
4.9 Conclusion
Important Websites
References
Part 2: Functionalized Carbon Nanotubes: Current and Emerging Biomedical Applications
Chapter 5 Functionalized Carbon Nanotubes: Applications in Biosensing
5.1 Introduction
5.2 CNTs-Based Biosensors
5.2.1 Electrochemical Biosensors
5.2.1.1 Electrochemical Enzyme Sensors
5.2.1.2 Electrochemical Immunosensors
5.2.1.3 Electrochemical DNA Sensors
5.2.1.4 Non-Biomolecule Based Electrochemical Sensors
5.2.2 Optical CNT Sensors
5.2.3 Field-Effect CNTs Sensors
5.2.4 CNT Human Strain Sensor
5.3 Conclusion
References
Chapter 6 Applications of Functionalized Carbon Nanotubes in Drug Delivery Systems
6.1 Introduction
6.2 Nanoparticles-Doped Carbon Nanotubes
6.3 Brain-Targeted Delivery
6.4 The Organic Molecules Functionalized CNTs as Drug Delivery Vehicles
6.5 Functionalized CNTs with Nanoparticles for Drug Active Molecular Mechanism
6.5.1 Future of Scope of Functionalized Carbon Nanotube Drug Delivery Application
6.6 Conclusion
References
Chapter 7 Functionalized Carbon Nanotubes for Gene Therapy
7.1 Introduction
7.2 Functionalized CNTs and Gene Therapy
7.3 Cellular Uptake of CNT
7.4 Functionalized Carbon Nanotubes and Cancer
7.5 Miscellaneous Diseases and Gene Delivery Through Functionalized CNT
7.6 Toxicology and Environmental Aspects of Functionalized CNT
7.6.1 Cellular Toxicity
7.6.2 Liver Toxicity
7.6.3 Central Nervous System Toxicity
7.6.4 Cardiovascular Toxicity
7.7 Regulatory Concerns Over Functionalized Carbon Nanotubes
7.8 Conclusion and Future Prospects
Important Website
References
Chapter 8 Applications of Functionalized Carbon Nanotubes in Cancer Therapy and Diagnosis
8.1 Introduction
8.2 Characteristic Properties of CNTs and Their Performance
8.2.1 Physicochemical Properties of CNTs
8.3 The Techniques of CNTs Functionalization
8.4 Application of Carbon Nanotubes in Cancer Therapy and Diagnostic
8.4.1 The Use of Carbon Nanotubes in Cancer Treatment
8.4.2 Intracellular Targeting Using Carbon Nanotubes
8.4.2.1 Nucleus Targeting
8.4.2.2 Cytoplasm Targeting
8.4.2.3 Mitochondria Targeting
8.4.3 CNTs for Immunotherapy
8.4.4 Cancer Stem Cell Inhibition
8.5 Carbon Nanotubes in Cancer Diagnosis
8.5.1 CNTs in Cancer Imaging
8.5.1.1 Raman Imaging
8.5.1.2 Nuclear Magnetic Resonance Imaging
8.5.1.3 Ultrasonography
8.5.1.4 Photoacoustic Imaging
8.5.1.5 Near‑Infrared Fluorescence Imaging
8.6 Future Prospects
8.7 Conclusion
Important Websites
References
Chapter 9 Functionalized Carbon Nanotubes for Biomedical Imaging: The Recent Advances
9.1 Introduction
9.2 CNT-Based Imaging Methods
9.2.1 Fluorescence Imaging
9.2.2 Raman Imaging
9.2.3 Photoacoustic Imaging
9.2.4 Magnetic Resonance Imaging
9.2.5 Nuclear Imaging
9.3 Prospects and Challenges
9.4 Conclusion
References
Chapter 10 Functionalized Carbon Nanotubes for Artificial Bone Tissue Engineering
10.1 Introduction
10.2 CNT-Based Scaffolds and Implants
10.2.1 Hydroxyapatite
10.2.2 Polymers
10.2.2.1 Poly(ε-Caprolactone)
10.2.2.2 Polymethyl-Methacrylate
10.2.2.3 Poly(Lactide-Co-Glycolide)
10.2.2.4 Poly-L-Lactic Acid
10.2.2.5 Polyvinyl Alcohol
10.2.2.6 Others
10.2.3 Biopolymers
10.2.3.1 Chitosan
10.2.3.2 Collagen
10.2.3.3 Others
10.3 Intellectual Property Rights and Commercialization Aspects
10.4 Conclusion and Future Perspectives
References
Chapter 11 Application of Functionalized Carbon Nanotubes in Biomimetic/Bioinspired Systems
11.1 Introduction
11.2 Naturally Occurring Materials
11.2.1 Nacre and Bone
11.2.2 Petal Effect and Gecko Feet
11.2.3 Lotus Effect
11.2.4 Structural Colors, Antireflection, and Light Collection
11.3 Bioinspired Functionalized CNTs Material
11.4 Challenges and Solutions in Using CNTs
11.5 Conclusion and Perspectives
References
Chapter 12 Functionalized Carbon Nanotubes: Applications in Tissue Engineering
12.1 Introduction
12.2 Structural, Physical, and Chemical Properties
12.3 Interactions and Biodegradation of CNTs with Biomolecule
12.4 Bio-Security of CNT-Based Scaffolds Toward In Vivo Analyses
12.5 CNTs Towards the Bone Compatibility
12.6 Applications of Functionalized CNTs in Tissue Engineering
12.6.1 Functionalized CNTs for Cardiac Tissue Engineering
12.6.2 Functionalized CNTs for Neuronal Tissue Regeneration
12.6.3 Functionalized CNT for Cartilage Tissue Engineering
12.6.4 CNT for Bone Tissue Regeneration
12.7 Future Perspectives and Challenges
12.8 Conclusion
Important Websites
References
Chapter 13 Functionalized Carbon Nanotubes for Cell Tracking
Abbreviations
13.1 Introduction
13.2 Carbon Nanotubes
13.2.1 Cellular Interaction of CNTs
13.3 Cellular Tracking via CNT
13.3.1 Effect of the Surface Coating of CNTs in Single-Particle Tracking
13.4 3D Tracking Using CNTs
13.4.1 Detection of Single Protein Molecules Through CNTs
13.4.2 Stem Cell Labeling and Tracking Through CNTs
13.4.3 Labelling and Tracking of Human Pancreatic Cells Through CNTs
13.4.4 CNT as Macrophage Carrying Microdevices
13.4.4.1 Intracellular Fluctuations and CNT
13.4.5 Limitations of CNTs
13.5 Concluding Remarks and Future Perspective
Important Links
Acknowledgment
References
Chapter 14 Functionalized Carbon Nanotubes for Treatment of Various Diseases
14.1 Introduction
14.2 CNTs: Basic Structure, and Synthesis Methods
14.2.1 Structure and Synthesis of CNTs
14.2.2 Arc Discharge Technique
14.2.3 Laser Ablation Technique
14.2.4 Catalytic Chemical Vapor Deposition Technique
14.3 Functionalization of CNTs
14.3.1 Covalent Functionalization
14.3.2 Non-Covalent Functionalization
14.4 Toxicity/Bio-Safety Profile of Carbon Nanotubes
14.5 Investigating the Promising Biomedical Effects of Functionalized CNTs
14.5.1 Functionalized CNTs-Based Remediation of Infectious Diseases
14.5.2 Functionalized CNTs for the Treatment of Central Nervous System Disorders (CNS)
14.5.3 Functionalized CNTs for Gene Delivery
14.5.4 Implication of Functionalized CNTs in Cancer Diagnosis and Treatment
14.5.5 Functionalized CNTs for Drug Targeting and Release
14.6 Future Prospective
14.7 Conclusion
Important Websites
References
Chapter 15 Role of Functionalized Carbon Nanotubes in Antimicrobial Activity: A Review
15.1 Introduction
15.2 Introduction to CNTs
15.2.1 Classification of CNTs
15.2.2 Structure of CNTs
15.3 Overview on CNTs Functionalization
15.3.1 Types of Functionalization
15.4 Anti-Microbial Activity of f-CNTs: Interaction and Action
15.5 Antifungal Activity of f-CNTs
15.6 Antibacterial Activity of f-CNTs
15.6.1 For SWNTs
15.6.2 For MWCNTs
15.7 Commercial Application of Antimicrobial Activity of f-CNTs
15.8 Overview on Antimicrobial Activity of f-CNTs
15.9 Future Scope
15.10 Conclusion
Acknowledgement
References
Index
EULA

Citation preview

Functionalized Carbon Nanotubes for Biomedical Applications

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Functionalized Carbon Nanotubes for Biomedical Applications

Edited by

Jeenat Aslam

Department of Chemistry, College of Science, Taibah University, Yanbu, Al-Madina, Saudi Arabia

Chaudhery Mustansar Hussain

Department of Chemistry & Environmental Science at the New Jersey Institute of Technology, Newark, NJ., U.S.A. and

Ruby Aslam

Department of Applied Chemistry, Aligarh Muslim University, U.P., India

This edition first published 2023 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2023 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­ resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­ tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­ tion does not mean that the publisher and authors endorse the information or services the organiza­ tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 978-1-119-90483-0 Cover image: Pixabay.Com Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xv

Part 1: Overview of Functionalized Carbon Nanotubes 1 1 Functionalized Carbon Nanotubes: An Introduction Sheerin Masroor 1.1 Introduction 1.2 Carbon Nanotube’s Classification 1.3 Structural and Morphological Analysis of Carbon Nanotubes 1.4 Synthetic Techniques of Carbon Nanotubes 1.5 Functionalization of Carbon Nanotubes 1.6 Commercial Scale Use of Functionalized Carbon Nanotubes 1.7 Conclusion and Future Prospects References 2 Functionalized Carbon Nanotubes: Synthesis and Characterization Neelam Sharma, Shubhra Pareek, Rahul Shrivastava and Debasis Behera 2.1 Introduction 2.2 Synthesis Methods 2.2.1 Arc Discharge 2.2.2 Laser Ablation 2.2.3 Chemical Vapor Deposition 2.3 Characterization 2.3.1 Raman Spectroscopy 2.3.2 Fourier Transform Infrared Spectroscopy (FT-IR) 2.3.3 Thermogravimetric Analysis (TGA) 2.3.4 Scanning Electron Microscopy (SEM) 2.3.5 Transmission Electron Microscopy (TEM) 2.3.6 X-Ray Diffraction (XRD)

3 4 6 7 8 9 12 14 15 21 22 24 24 25 26 27 27 28 29 29 30 31 v

vi  Contents 2.3.7 X-Ray Photoelectron Spectroscopy (XPS) 2.4 Functionalized Routes of CNTs 2.4.1 Surface Oxidation 2.4.2 Doping Heteroatoms 2.4.3 Alkali Activation 2.4.4 Sulfonation 2.4.5 Halogenation 2.4.6 Grafting 2.4.6.1 Grafting via Oxygen-Containing Groups 2.4.6.2 Grafting via Diazonium Compounds 2.4.6.3 Other Grafting Methods 2.4.7 Non-Covalent Functionalization of CNTs 2.4.8 Deposition on Functionalized CNTs 2.4.9 Physiochemical Approaches 2.4.10 Electrochemical Deposition 2.4.11 Electroless Deposition 2.5 Conclusion References

32 33 33 33 33 34 34 34 35 36 37 37 37 38 38 39 39 40

3 Carbon Nanotubes: Types of Functionalization 49 Manilal Murmu, Debanjan Dey, Naresh Chandra Murmu and Priyabrata Banerjee 3.1 Introduction 50 3.2 Carbon Nanotubes 50 3.3 Functionalization of Carbon Nanotubes 52 3.3.1 Covalent Functionalization 52 3.3.2 Non-Covalent Functionalization of Carbon Nanotubes 58 3.3.2.1 Reversibility in Non-Covalent Functionalization 63 3.3.2.2 Solvent Variation in Non-Covalent Functionalization 64 3.3.3.3 pH of the System in Non-Covalent Functionalization 64 3.3.3.4 Temperature Responsive System in Non-Covalent Functionalization 65 3.4 Conclusion and Future Outlook 65 Acknowledgements 65 Web Links 66 References 66

Contents  vii 4 Functionalization Carbon Nanotubes Innovate on Medical Technology 75 Afroz Aslam, Jeenat Aslam, Hilal Ahmad Parray and Chaudhery Mustansar Hussain 4.1 Introduction 75 4.2 Functionalization CNTs for Biomedical Applications 78 4.3 Potential Applications of CNTs in Cancer Therapy 79 4.3.1 Anti-Tumor Immunotherapy 80 4.3.2 Anti-Tumor Hyperthermia Therapy 80 4.3.3 Anti-Tumor Chemotherapy 81 4.3.4 Other Cancer Treatment Strategies 82 4.4 Treatment of Central Nervous System Disorders 82 4.5 Treatment of Infectious Diseases 84 4.6 CNTs-Based Transdermal Drug Delivery 85 4.7 f-CNTs for Vaccination 86 4.8 Application of f-CNTs in Tissue Engineering 86 4.9 Conclusion 88 Important Websites 89 References 89

Part 2: Functionalized Carbon Nanotubes: Current and Emerging Biomedical Applications

95

5 Functionalized Carbon Nanotubes: Applications in Biosensing 97 N. Palaniappan, Nidhi Vashistha and Ruby Aslam 5.1 Introduction 97 5.2 CNTs-Based Biosensors 99 5.2.1 Electrochemical Biosensors 100 5.2.1.1 Electrochemical Enzyme Sensors 100 5.2.1.2 Electrochemical Immunosensors 101 5.2.1.3 Electrochemical DNA Sensors 102 5.2.1.4 Non-Biomolecule Based Electrochemical Sensors 104 5.2.2 Optical CNT Sensors 105 5.2.3 Field-Effect CNTs Sensors 106 5.2.4 CNT Human Strain Sensor 107 5.3 Conclusion 108 References 108

viii  Contents 6 Applications of Functionalized Carbon Nanotubes in Drug Delivery Systems N. Palaniappan, Małgorzata Kujawska and Kader Poturcu 6.1 Introduction 6.2 Nanoparticles-Doped Carbon Nanotubes 6.3 Brain-Targeted Delivery 6.4 The Organic Molecules Functionalized CNTs as Drug Delivery Vehicles 6.5 Functionalized CNTs with Nanoparticles for Drug Active Molecular Mechanism 6.5.1 Future of Scope of Functionalized Carbon Nanotube Drug Delivery Application 6.6 Conclusion References 7 Functionalized Carbon Nanotubes for Gene Therapy Tejas Agnihotri, Tanuja Shinde, Manoj Gitte, Pankaj Kumar Paradia, Rakesh Kumar Tekade and Aakanchha Jain 7.1 Introduction 7.2 Functionalized CNTs and Gene Therapy 7.3 Cellular Uptake of CNT 7.4 Functionalized Carbon Nanotubes and Cancer 7.5 Miscellaneous Diseases and Gene Delivery Through Functionalized CNT 7.6 Toxicology and Environmental Aspects of Functionalized CNT 7.6.1 Cellular Toxicity 7.6.2 Liver Toxicity 7.6.3 Central Nervous System Toxicity 7.6.4 Cardiovascular Toxicity 7.7 Regulatory Concerns Over Functionalized Carbon Nanotubes 7.8 Conclusion and Future Prospects Important Website References

117 118 121 123 125 126 126 127 127 139

140 141 146 147 150 158 159 159 160 161 162 164 165 165

Contents  ix 8 Applications of Functionalized Carbon Nanotubes in Cancer Therapy and Diagnosis Irshad Ahmad, Talat Parween, Lina Khandare, Aafaq Tantray and Weqar Ahmad Siddiqi 8.1 Introduction 8.2 Characteristic Properties of CNTs and Their Performance 8.2.1 Physicochemical Properties of CNTs 8.3 The Techniques of CNTs Functionalization 8.4 Application of Carbon Nanotubes in Cancer Therapy and Diagnostic 8.4.1 The Use of Carbon Nanotubes in Cancer Treatment 8.4.2 Intracellular Targeting Using Carbon Nanotubes 8.4.2.1 Nucleus Targeting 8.4.2.2 Cytoplasm Targeting 8.4.2.3 Mitochondria Targeting 8.4.3 CNTs for Immunotherapy 8.4.4 Cancer Stem Cell Inhibition 8.5 Carbon Nanotubes in Cancer Diagnosis 8.5.1 CNTs in Cancer Imaging 8.5.1.1 Raman Imaging 8.5.1.2 Nuclear Magnetic Resonance Imaging 8.5.1.3 Ultrasonography 8.5.1.4 Photoacoustic Imaging 8.5.1.5 Near‑Infrared Fluorescence Imaging 8.6 Future Prospects 8.7 Conclusion Important Websites References 9 Functionalized Carbon Nanotubes for Biomedical Imaging: The Recent Advances Alina Abbas, Saman Zehra, Ruby Aslam, Mohammad Mobin and Shahidul Islam bhat 9.1 Introduction 9.2 CNT-Based Imaging Methods 9.2.1 Fluorescence Imaging 9.2.2 Raman Imaging

171 172 175 176 177 180 180 180 181 181 181 182 183 183 184 184 184 184 185 185 186 186 187 188 197 198 199 200 204

x  Contents 9.2.3 Photoacoustic Imaging 9.2.4 Magnetic Resonance Imaging 9.2.5 Nuclear Imaging 9.3 Prospects and Challenges 9.4 Conclusion References 10 Functionalized Carbon Nanotubes for Artificial Bone Tissue Engineering Sougata Ghosh and Ebrahim Mostafavi 10.1 Introduction 10.2 CNT-Based Scaffolds and Implants 10.2.1 Hydroxyapatite 10.2.2 Polymers 10.2.2.1 Poly(ε-Caprolactone) 10.2.2.2 Polymethyl-Methacrylate 10.2.2.3 Poly(Lactide-Co-Glycolide) 10.2.2.4 Poly-L-Lactic Acid 10.2.2.5 Polyvinyl Alcohol 10.2.2.6 Others 10.2.3 Biopolymers 10.2.3.1 Chitosan 10.2.3.2 Collagen 10.2.3.3 Others 10.3 Intellectual Property Rights and Commercialization Aspects 10.4 Conclusion and Future Perspectives References 11 Application of Functionalized Carbon Nanotubes in Biomimetic/Bioinspired Systems Mohammad Mobin, Ruby Aslam, Saman Zehra, Jeenat Aslam and Shahidul Islam bhat 11.1 Introduction 11.2 Naturally Occurring Materials 11.2.1 Nacre and Bone 11.2.2 Petal Effect and Gecko Feet 11.2.3 Lotus Effect 11.2.4 Structural Colors, Antireflection, and Light Collection 11.3 Bioinspired Functionalized CNTs Material

207 209 212 212 214 214 225 226 230 231 234 235 237 238 240 241 242 242 244 244 247 248 251 252 257 258 259 259 259 260 261 261

Contents  xi 11.4 Challenges and Solutions in Using CNTs 11.5 Conclusion and Perspectives References

272 272 274

12 Functionalized Carbon Nanotubes: Applications in Tissue Engineering 281 Ajahar Khan, Khalid A. Alamry and Raed H. Althomali 12.1 Introduction 282 12.2 Structural, Physical, and Chemical Properties 284 12.3 Interactions and Biodegradation of CNTs with Biomolecule 287 12.4 Bio-Security of CNT-Based Scaffolds Toward In Vivo Analyses 288 12.5 CNTs Towards the Bone Compatibility 293 12.6 Applications of Functionalized CNTs in Tissue Engineering 294 12.6.1 Functionalized CNTs for Cardiac Tissue Engineering 294 12.6.2 Functionalized CNTs for Neuronal Tissue Regeneration 297 12.6.3 Functionalized CNT for Cartilage Tissue Engineering 298 12.6.4 CNT for Bone Tissue Regeneration 300 12.7 Future Perspectives and Challenges 303 12.8 Conclusion 304 Important Websites 305 References 305 13 Functionalized Carbon Nanotubes for Cell Tracking Sagar Salave, Dhwani Rana, Jyotsna Vitore and Aakanchha Jain Abbreviations 13.1 Introduction 13.2 Carbon Nanotubes 13.2.1 Cellular Interaction of CNTs 13.3 Cellular Tracking via CNT 13.3.1 Effect of the Surface Coating of CNTs in Single-Particle Tracking 13.4 3D Tracking Using CNTs 13.4.1 Detection of Single Protein Molecules Through CNTs

319 319 320 321 325 325 328 328 329

xii  Contents 13.4.2 Stem Cell Labeling and Tracking Through CNTs 13.4.3 Labelling and Tracking of Human Pancreatic Cells Through CNTs 13.4.4 CNT as Macrophage Carrying Microdevices 13.4.4.1 Intracellular Fluctuations and CNT 13.4.5 Limitations of CNTs 13.5 Concluding Remarks and Future Perspective Important Links Acknowledgment References 14 Functionalized Carbon Nanotubes for Treatment of Various Diseases Ajahar Khan, Khalid A. Alamry and Raed H. Althomali 14.1 Introduction 14.2 CNTs: Basic Structure, and Synthesis Methods 14.2.1 Structure and Synthesis of CNTs 14.2.2 Arc Discharge Technique 14.2.3 Laser Ablation Technique 14.2.4 Catalytic Chemical Vapor Deposition Technique 14.3 Functionalization of CNTs 14.3.1 Covalent Functionalization 14.3.2 Non-Covalent Functionalization 14.4 Toxicity/Bio-Safety Profile of Carbon Nanotubes 14.5 Investigating the Promising Biomedical Effects of Functionalized CNTs 14.5.1 Functionalized CNTs-Based Remediation of Infectious Diseases 14.5.2 Functionalized CNTs for the Treatment of Central Nervous System Disorders (CNS) 14.5.3 Functionalized CNTs for Gene Delivery 14.5.4 Implication of Functionalized CNTs in Cancer Diagnosis and Treatment 14.5.5 Functionalized CNTs for Drug Targeting and Release 14.6 Future Prospective 14.7 Conclusion Important Websites References

330 330 331 331 332 332 333 333 333 339 340 342 342 342 342 343 343 344 344 346 349 350 350 351 354 357 362 363 364 365

Contents  xiii 15 Role of Functionalized Carbon Nanotubes in Antimicrobial Activity: A Review Monika Aggarwal, Samina Husain and Basant Kumar 15.1 Introduction 15.2 Introduction to CNTs 15.2.1 Classification of CNTs 15.2.2 Structure of CNTs 15.3 Overview on CNTs Functionalization 15.3.1 Types of Functionalization 15.4 Anti-Microbial Activity of f-CNTs: Interaction and Action 15.5 Antifungal Activity of f-CNTs 15.6 Antibacterial Activity of f-CNTs 15.6.1 For SWNTs 15.6.2 For MWCNTs 15.7 Commercial Application of Antimicrobial Activity of f-CNTs 15.8 Overview on Antimicrobial Activity of f-CNTs 15.9 Future Scope 15.10 Conclusion Acknowledgement References

377 378 378 379 381 382 384 387 388 390 390 392 400 401 405 405 406 406

Index 413

Preface Nanotechnology suggests fascinating opportunities for a variety of applications in the biomedical field, including bioimaging and targeted delivery of biomacromolecules to cells. Numerous strategies have been recommended to functionalize carbon nanotubes with raised solubility for efficient use in these biomedical applications. Functionalized carbon nanotubes are relatively flexible, interact with cell membranes, and penetrate different biological tissues. As they possess unique arrangements and extravagant mechanical, thermal, magnetic, optical, electrical, surface, and chemical properties—the combination of which gives them widespread biomedical applications—considerable effort has been made to employ them as new materials for the development of novel biomedical tools, such as diagnostic sensors, imaging agents, and drug/gene delivery systems for both diagnostics and clinical treatment. Therefore, tremendous progress continues to be made in this field, as is reflected in the rapid growth in the amount of scattered literature available. This book summarizes the recent progress and developments in functionalized carbon nanotubes-based biomedical devices and systems at both the experimental and theoretical model scale. It covers a broad range of topics relating to carbon nanotubes, from synthesis and functionalization to applications in advanced biomedical devices and systems. The book is logically and eloquently designed to capture an inclusive picture of functionalized carbon nanotubes-based biomedical devices and systems, thereby offering the reader a concentrated up-to-date reference source. It is divided into two parts. An overview of functionalized carbon nanotubes is presented in Part 1, in which Chapter 1 introduces functionalized carbon nanotubes and Chapter 2 describes their synthesis and characterization. Then various types of functionalization of carbon nanotubes are summarized in Chapter 3, after which their role in innovative medical technology is explored in Chapter 4. Different current and emerging biomedical applications of functionalized carbon nanotubes are covered in Part 2, beginning with Chapter 5, which discusses functionalized carbon nanotubes in xv

xvi  Preface biosensing. Then their use in drug delivery systems and gene therapy are covered in Chapters 6 and 7, respectively. The applications of functionalized carbon nanotubes in cancer therapy and diagnosis are discussed in Chapter 8, and recent advances in their use for biomedical imaging in Chapter 9. Functionalized carbon nanotubes for bone tissue engineering are discussed in Chapter 10, and their use in biomimetic/bioinspired systems in Chapter 11. Finally, Chapters 12 to 15 discuss the application of functionalized carbon nanotubes in tissue engineering, cell tracking, disease treatment, and antimicrobial activity, respectively. The aim of this book is to convey information on the recent advancements in the functionalized carbon nanotubes-based biomedical devices and systems arena. It is intended for a very wide-ranging audience working in the fields of advanced materials science, chemistry, medical engineering technology, etc. Since the chapters were contributed and edited by renown researchers, scientists and experts from academia and industry, this book will be an invaluable reference addition to any library collection, including that of universities, industrial institutions, government and independent institutes, and individual research groups and scientists. Overall, it was designed to be useful for advanced undergraduate or graduate students, researchers and scientists who are searching for the functionalized carbon nanotubes-based biomedical devices and systems that modern research demands. We are very grateful to all those whose exceptional efforts made this book possible. Special thanks go to Mr. Martin Scrivener and the editorial team at Scrivener Publishing for their wholehearted support throughout this project, and to Wiley-Scrivener for publishing the book. The Editors: Jeenat Aslam Chaudhery Mustansar Hussain Ruby Aslam

Part 1 OVERVIEW OF FUNCTIONALIZED CARBON NANOTUBES

1 Functionalized Carbon Nanotubes: An Introduction Sheerin Masroor

*

Department of Chemistry, A. N. College, Patliputra University, Patna, Bihar, India

Abstract

Carbon nanotubes written in short form as, CNTs are the tubes which are made from carbon having diameters in nanometers (nm) or 10-9 meters. They can be considered as one of the best carbon allotropes like graphene, graphite, fullerene, diamond and amorphous carbon. Many experimental processes have been obtained to synthesize nanotubes in different sizeable quantities, such as chemical vapor deposition, arc discharge, and laser ablation methods. The blooming of technology related to nanomaterials is mostly happened in drug delivery, biomedical imaging, biosensing and designing of useful nanocomposites. While some more methods relating and realizing applications are continuing to evolve. Carbon nanotubes may be of two types single-wall carbon nanotubes (SWCNTs) having diameters in the range of a nanometers only or multi-wall carbon nanotubes (MWCNTs) possesses nest like structure of single-wall carbon nanotubes. These tubes can be allowed to functionalize via two general reactions such as esterification and amidation of nanotubes with carboxylic acids ends in it. General property like solubility of these tubes helps to know the properties of them by applying solution-based processes. A number of literatures relating functionalization of the carbon nanotubes in the modification of applications in nanocomposites and biological techniques can be seen there. Keywords:  Carbon nanotubes, allotropes, nanocomposites, drug, esterification

Email: [email protected]

*

Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (3–20) © 2023 Scrivener Publishing LLC

3

4  Functionalized CNT for Biomedical Applications

1.1 Introduction Carbon (C) is having atomic number six (6), which is non-metallic in nature and having (four electrons or tetravalency available to form covalent chemical bonds and makes about 0.025 percent of Earth’s crust [1, 2]. There are three naturally occurring isotopes of carbon discovered so far, symbolized as 12C and 13C and 14C. Out of all three first two are stable while third one is radionuclide whose half-life is about 5,730 years [3]. Also, the carbon is considered as 15th most existed element in the crust of Earth, and the fourth most abundant element in the universe in terms of mass after hydrogen (H2), helium (He), and oxygen (O2). Carbon is abundant element found and its unique diversity to form multiple organic compounds or collection of monomers (polymers) at the temperatures commonly encountered on Earth surface enables this element to serve as a the most common element of all known life. In addition, it is also considered as the second most abundant element found in the human body by mass of approximately 18.5% after oxygen [4]. It’s a unique property of carbon atoms that they can bind together in multiple forms generating different number of carbon allotropes. Some of the well-defined allotropes of carbon include graphite, amorphous carbon, fullerenes and diamond. In turn the physical property of carbon mainly depends upon its allotropic form, like graphite is black in color and opaque, while diamond is transparent in nature. Graphite is known for its softness while the diamond is hardest material. In terms of electrical conductivity graphite always acts as good electrical conductor while diamond has diminished electrical conductivity. Recently carbon nanotubes have been also studied for best thermal conductivities along with graphite, graphene and diamond at standard temperature and pressure (STP) or under normal conditions. It can be considered as most of the carbon allotropes are solid at STP or under normal conditions, while graphite is being effectively stable thermodynamically at STP. They all are resistant chemically and generally require very high temperature and oxygen to make reaction. The carbon in atomic form is very short-lived in nature and hence it is stabilized in different multi-atomic structures with vast molecular arrangements which are generally called as allotropes. One allotrope of carbon which is fullerene is commonly synthesized nowadays successfully and uniquely harvesting it in research in the form of carbon nanotubes, carbon nanobuds, bucky balls, and nanofibers [5–10].

Introduction: Functionalized Carbon Nanotubes  5 Diverse variety of the exotic allotropes has also been discovered so far, such as glassy carbon, lonsdaleite, carbon nanofoam, and linear acetylenic carbon (carbyne) [11–14]. Two natural occurring crystalline forms of pure carbon are graphite and diamond. Here in diamond, the atoms of carbon show sp3 hybridization wherein the four bonds are directly attached towards the corners of regular tetrahedron, making diamond so strong and rigid due to three-dimensional network. While in graphite we can easily see carbons hybridization in sp2 form, in which all atoms are joined uniformly to three carbons with an angle of 120°. In 1985 a new form of carbon, Buckminster fullerene symbolized as (C60) was discovered by Korto et al. [15]. In addition, with graphite, diamond and fullerene (C60), quasi-one-dimensional nanotube was also invented in another form of carbon which was first reported in 1991 by Ijima. He reported in his finding that the soot was made by an arc-discharge method to synthesize multiwalled carbon nanotube-MWCNTs [16]. One of the allotropes of carbon is which may be tubular or rod like structure in shape fully made of graphite. The provided tubes may have at least two to many layers ranged for diameter from 3 to 30 nm in size. Later in about two years, the single-walled carbon nanotubes (SWCNTs) came into existence [17]. In and around same period of time, Dresselhaus and his co-workers synthesized single-walled carbon nanotubes by following the previous track of producing multi walled carbon nanotubes (MWCNTs) with the addition of some transition metal particles to the electrodes of carbon [18]. The expected shapes of single-walled carbon nanotubes and multiple walled carbon nanotubes can be depicted from the Figure 1.1 (a and b). Here we can easily see that wall of SWCNT are

(a)

(b)

Figure 1.1  Structure of carbon nanotube (a). Single walled carbon nanotubes (b). Multiple walled carbon nanotubes.

6  Functionalized CNT for Biomedical Applications

Material Science

Drug Delivery System

Utilization of Functionalized Carbon Nanotubes

Water purif ications

Biomedical Science

Figure 1.2  Important applications of functionalized CNTs.

generally narrower than the MWCNT, which is having a diameter in the range of 1–2 nm, and took curved shape instead to be straight. The utmost properties of carbon nanotubes such as electrical, electrochemical, mechanical and chemical properties are extensively studied by huge amount of research that has been going on throughout since last few decades. Various reactions had been going on to find best possible results to harvest them at best possible. Nowadays the researchers have been focusing on changing/improving the quality of nanotubes those who are engaged in catalytic reactions [19]. Some important utilization of functionalized CNTs is pictorially present here in Figure 1.2.

1.2 Carbon Nanotube’s Classification Broadly classified in the given ways such as [20–23]: a. Single-walled carbon nanotubes (SWCNTs). b. Multiple-walled carbon nanotubes (MWCNTs). a. Single-walled carbon nanotubes (SWCNTs). • They are only made from single layer of graphene. • The synthetic route requires catalyst, which requires an appropriate control over growth and reaction conditions. • Can exist in bundle structures. • If synthesized the product may be found with the yield percentage of 30-50% by general method but if synthesized via arc discharged synthetic method, yield may be achieved up to 80%.

Introduction: Functionalized Carbon Nanotubes  7

b.

c.

d.

e.

• As they are simple in nature, they are easy to characterize on synthesis. Multiple-walled carbon nanotubes (MWCNTs). • They are made from multiple layers of graphene. • Synthetic route doesn’t need catalyst and bulk synthesis is easy. • The purity is high by the synthetic route of Chemical vapor deposition (CVD) method with a yield of about 35–90%. • Here the unintentional defect is less especially when it is synthesized by arc-discharged method. • It has a convoluted structure which can’t be easily twistable. Armchair Carbon Nanotubes: It is a kind of single-walled carbon nanotube that has equal n and m indices and mainly depends on how the graphite is “rolled up” during its synthetic process. Here in this type of carbon nanotube, chiral angle is 30° and can be considered as metallic. This structure has been manually explained by the ChEBITeam [24]. Zigzag Carbon Nanotubes: The zigzag nanotubes are having n,0 indices and are metallic in nature only and only if the index n is a multiple of 3. Specifically, we can say one-third of zigzag carbon nanotubes are metallic in nature, while rest two-thirds are semiconductors [25]. Chiral Carbon Nanotubes: A carbon nanotube is said to be chiral if it has (n,m) type of indices where m > 0 and m ≠ n; and its enantiomer (mirror image) has type (m,n), which is different from (n,m).

1.3 Structural and Morphological Analysis of Carbon Nanotubes The only component present in single walled carbon nanotubes (SWCNTs) is carbon only which can be seen as tubular shells which are rolled up in

8  Functionalized CNT for Biomedical Applications the form of graphene sheets, especially benzene (Cyclohexa-1,3,5-triene) type hexagonal rings of carbon atoms. These graphene sheets look cylinders which may be borrowed from a honeycomb lattice, showing a single atomic layer of crystalline graphite. While the multiwalled carbon nanotubes are heap of graphene sheets which can be rolled up into cylinders in the concentric form. Nanotube can be considered as single molecule made up of millions of atoms and whose length can be in the range of micrometers with diameters of about 0.7 nanometers [26]. The single walled carbon nanotubes mainly contain ten (10) atoms which are present over the circumference whose thickness is only one atom thick of the whole tube. Generally, the carbon nanotubes have massive length-to-diameter ratio of approx. value of about 1000, which conclude them as one-dimensional structures [27]. The structure of multi walled carbon nanotube, mainly contains large and numerous single walled tubes which are stacked over and above each other. If the diameter of nanostructures formed have a diameter of about 15 nanometers, then it can be called as multi walled carbon nanotubes otherwise the structures are better known as carbon nanofibers, which are strands of layered-graphite sheets [28].

1.4 Synthetic Techniques of Carbon Nanotubes There is a great demand of high-quality nanotube materials which can be used for fundamental and technological applications. The quality index can be measured on the basis of presence or absence of chemical and structural defects over a notable scale length of 1-10 microns along the axes of tube. In last few decades it can be easily seen from the literature review that research publication in the form of papers, reviews, book chapters or patents have been increasing enormously. Although there are also so many complications and challenges to think for and must be to resolve relating synthetic procedures of carbon nanotubes. On a broader vision, some existing challenges in the era of nanotube synthetic routes are: large scale production of carbon nanotubes with best quality, Controlled production over specific structure and their properties, development of distinct mechanism via thorough study of the processes of nanotube’s growth. In lieu of these numerous techniques have been developed to synthesize carbon nanotubes with discrete morphology and structures in the research laboratory. At present there are specifically three synthetic routes generally used to amalgamate carbon nanotubes and these are chemical vapor deposition, arc discharge and laser ablation methods [29–35].

Introduction: Functionalized Carbon Nanotubes  9 The important ingredients to synthesize carbon nanotubes are a pure carbon, catalyst and adequate amount of energy. The procedure followed mainly have a step of adding ample energy to a carbon which can be able to produce a fragment which then recombine in their own way to create carbon nanotube. The source of energy may be of any kind like electricity from an arc discharge or heat from a furnace with a temperature of approx. 900°C) or the high-intensity light from laser ablation. The comparison between three processes is given below: Arc Discharge Method In this process where there is connection two pure graphite rods which supplies the power and put them at some millimeters apart. On passing 100 amps, and at low pressure inert gas like (Helium) the carbon vaporizes and hot plasma appears there. Single walled carbon nanotubes synthesized are mainly short tubes with diameters of about 0.6–1.4 nm while multiple carbon nanotubes formed are having inner diameter of about 1–3 nm and outer diameter with value 10 nm. One disadvantage of this method is that, it involves high cost with yield of 30–90%. Laser Ablation Method The process involves use of blast graphite amidst intense laser pulses and in the presence of argon/nitrogen gas at 500 Torr to give rise to carbon gas from which the carbon nanotubes will be formed. Major product involves formation of single walled carbon nanotubes with long bundles of tubes having length of 5–20 microns and individual diameter with value 1–2 nm. Synthesis involves high cost with yield of about 70%. Chemical Vapor Deposition Method Specifically, there is use of fossil-based hydrocarbon and botanical hydrocarbons and placing them in oven at high heat, further adding slowly a ­carbon-bearing gas like methane in it. As soon as gas decomposes it releases the carbon atoms attached to them which recombines to form nanotubes. The temperature applied is very high with value of 500–1000°C at atmospheric pressure. The yield ranges from 20–100%.

1.5 Functionalization of Carbon Nanotubes Upon synthesizing the nanotubes, we get them in large number differing in diameters and chirality’s. The reason behind is the presence of impurities in the form of metal and amorphous materials. This all requires chemical processing protocols post synthesis, which mainly helps them to divide carbon nanotubes on the basis of diameter and specific chirality [36, 37].

10  Functionalized CNT for Biomedical Applications The process of differentiating them depends mainly on electronic and mechanical properties of carbon nanotubes [38, 39]. The carbon nanotubes reinforcement efficiency is confined and severely confined because of its bad interfacial interaction, van der Waals interactions in between carbon nanotubes and polymer matrix. The dispersion of carbon nanotubes is very difficult from rest of the traditional fillers like as spherical particles and carbon fibers, due to their small diameter in nanoscale with high aspect ratio of greater than thousandth (1000th) and thus possessing large surface area. Furthermore, the commercialized form of carbon nanotubes is supplied in the form of heavily knotted bundles, causing difficulties in dispersion. To settle all problems, it was presumed earlier to develop some methods which can alter the surface properties of carbon nanotubes. This is called as functionalization of carbon nanotubes. These methods mainly divided into two divisions depending upon the interactions in between the carbon nanotubes and active materials: a. Covalent (Chemical) Functionalization, b. Noncovalent (Physical) Functionalization. a.  Covalent (Chemical) Functionalization, It is presumed that the end caps of carbon nanotubes favored to be made up of remarkably curved fullerene-like hemispheres, so they are therefore highly reactive, comparable to side walls [40, 41]. In addition, sidewalls themselves have certain defect sites like sp3-hybridized defects, ­pentagon-heptagon pairs called Stone-Walls defects, and formed void in the nanotube lattice [42]. Some defects shown in single walled carbon nanotubes are discussed here: • Presence of five to seven membered rings in the carbon network, rather having normal six membered ring, which ultimately leads to a bent site in the tube. • Damage caused by oxidative conditions, leaving behind a hole at -COOH groups. • The sp3 hybridized defects happen. • Free/open end of single walled carbon nanotubes which mainly ends with -COOH, -NO2, -OH, -H, and =O groups. The covalent functionalization is basically a result of chemical reaction which leads to the formation of covalent bond between functional groups adjacent to carbon of nanotubes. It can be successfully performed at the

Introduction: Functionalized Carbon Nanotubes  11 end caps of nanotubes or at their sidewalls which have any defects over that place. The direct covalent sidewall functionalization can be related to the switching of hybridization from sp2 to sp3 and a continuous loss of p-conjugation system on graphene layer. This present process can be successfully made by reaction with some atoms or molecules of a high chemical reactivity. Some approaches for functionalization include fluorination, hydrogenation, cycloaddition (Diels-Alder reaction), chlorination, bromination, carbene, and nitrene addition and azomethineylides [43–51]. The next method to functionalize the carbon is defect functionalization. The intrinsic defects are added by oxidative damage to the nanotube network by strong acids which assent holes functionalized with oxygenated functional groups. Specifically speaking this method relates with the reaction of carbon nanotubes with strong acid such as nitric acid (HNO3), sulfuric acid (H2SO4), hydrochloride (HCl) or a mixture of any of them, or using strong oxidants like potassium permanganate (KMnO4), reactive plasma or ozone which tends to unlatched these tubes and to eventually generating oxygenated functional groups such as carboxylic acid or ketone or alcohol or ester groups, that serve to restrain many dissimilar types of chemical components onto the ends and defect sites of these carbon nanotubes. One advantage of functionalizing carbon nanotubes by the covalent methods is that it has good solubility in various organic solvents as the carbon nanotubes have different functional groups with polar or non-polar properties [52–62]. In addition, these approaches have two utmost drawbacks. Firstly, during the process of functionalization reaction, chiefly when applying the ruinous ultrasonication process, a numerous number of defects are mostly created on the carbon nanotubes sidewalls, and in few cases, tubes are fractured into smaller pieces. This all extremely affects the properties of carbon nanotubes and also disrupts the π-electron system in tubes affecting the thermal and electrical properties. Secondly the use of concentrated acids or strong oxidants to functionalize the carbon nanotube can create hazardous effects to the environment producing poisonous by products. b.  Non-covalent (Physical) functionalization The positive point to note for this non-covalent functionalization is that it may not destroy the conjugated system of the nanotubes sidewalls, and hence it does not affect the basic structural properties of the provided material. This method is a secondary method for modulating the interfacial properties of carbon nanotubes. The CNTs are functionalized non-­covalently by number of compounds such as surfactants, aromatic compounds, polymers,

12  Functionalized CNT for Biomedical Applications employing π-π stacking or any hydrophobic interactions for the rest of the part. In this approach the modification of nanotubes happens with the preservation of desired properties and also altering their solubilities quite exceptionally. The molecules such as polymer, biopolymers, surfactants and aromatic molecules (pyrene/porphyrin) and their derivatives on interacting with the sidewalls of nanotubes via π-π stacking interactions opened the way for the non-covalent functionalization. Number of researchers does this in their own ways like one reported a simple approach to the non-covalent functionalization of nanotubes sidewalls and the ensuing immobilization of biological molecules onto carbon nanotubes with a highest degree of control and specificity. Hecht et al. modified fabricated CNTs/FET devices with a molecule of zinc porphyrin derivative, which used to detect directly a photo induced electron transferring within the zinc porphyrin derivative-carbon nanotubes system [63, 64]. The conjugated polymers, are shown to serve as remarkable wrapping materials for the application of non-covalent functionalization of nanotubes which results π-π stacking and van-der Waals interactions between the conjugated polymer having aromatic rings and the exposed surfaces of carbon nanotubes [65–70].

1.6 Commercial Scale Use of Functionalized Carbon Nanotubes The carbon nanotubes can be considered as big source to elevate the economic development as out of many of them can be effectively commercialized. Some of them have been discussed here: a. Biomedical application. Here the target drugs are clubbed with carbon nanotube molecules for better results, the ultimate property of CNTs and their active counterparts to invade into deeper biological cells which further makes them interesting transportation for the transmission of small molecule drugs. Furthermore, the ability to carry one or multiple remedial agents are marvelous such as MWCNT-Taxanes [71], Hydroxyapatite– carbon nanotube (CNT) composites Hydroxyapatite drug [72] Poly-l-lactic acid matrix MWCNT and Poly-l-lactic acid matrix [73].

Introduction: Functionalized Carbon Nanotubes  13 b. Delivery target with CNTs. CNTs are employed for targeted delivery of proteins, nucleic acids, drugs, antibodies, and other agents to their respective sites of action. They are best applied in treatment of malignant disorders, which include choriocarcinoma, carcinoma of the cervix, breast cancer, prostate cancer, brain gliomas, and testicular tumors [74]. Furthermore, the functionalization of chitosan molecule on their surface speedup the cell attachment to the sidewalls of the carbon nanotubes, giving desired targeted release to the cells, and changing the drug absorption. Such systems have significant potential for the delivery of drugs, peptides, and nucleic acids. c. Nerve and Tissue Regeneration via CNT. The application of CNTs as reinforcing agents has been mainly attributed to their excellent and unique electrical and mechanical properties. CNTs may be the best tissue engineering candidate among numerous other materials of natural or synthetic origin for tissue scaffolds. In addition, they are less dense, highly flexible, and have a very high young’s modulus representing good stiffness. These properties are utilized to make lighter scaffolds with very high strength. It has also been shown by various researchers that the material surface-free energies also play an important role in influencing cell adhesion, leading to greater tissue regeneration. Surface functionalization of these CNTs further imparts to them biocompatibility and biodegradability, ideal for their use in the body. d. Vaccine delivery via CNT. Difficulty in absorption, hypersensitivity and anaphylactic reactions are some common drawbacks related to delivery of vaccine on target molecules. Out of many approaches produced so far, CNTs have been applied to vaccine delivery and the action was found to be improved [75]. Also, with this when CNTs are allowed to conjugate with antigenic peptides, can produce new system for safe and effective delivery of synthetic vaccines. e. Infectious Diseases Treatment via CNTs. Some infectious diseases, like flu (bird, avian or swine), tuberculosis, leishmaniasis, and other acute respiratory

14  Functionalized CNT for Biomedical Applications syndrome have always been a critical public health problem encountered within globe. The pathogens responsible for these diseases show’s a remarkable level of resistance against numerous antivirus and antibacterial drugs. Nowadays, the functionalized CNTs are showing great results in the treatment of various diseases because of the ability which allows easy conjugation with drugs such as dapsone and amphotericin B. [76, 77]. This all conjugation mainly shows reduced toxicity and elevated antimycotic efficiency. In addition, the CNTs own their own have antimicrobial activity because bacteria, like Escherichia coli may be absorbed onto the surface of the CNTs. The CNT may cause induce oxidation of the intercellular oxidant glutathione, ensuing increased oxidative stress on the bacterial cells and thus eventually cellular death. f. Diagnosis of Diseases via CNTs. The biosensors have been in use since many decades in the field of diagnostics which have made a gigantic impact on basic scientific research and healthcare. These have quality to detect chemical, physical or biological quantity and transience it in the form of a signal. Properties such as fast electron transfer rate, big potential window, flexible surface chemistry, and good biocompatibility of carbon nanotubes can give them excellent property for their special use in biosensors. Recently, the research has disclosed the fact that CNTs can also generate quantum dots. QDs, or may act like quantum dots. Because of the light-emitting property, these molecules have potential applications in taking imaging of various body parts [78].

1.7 Conclusion and Future Prospects As in the recent years development mainly based on the advancement of material science and nanotechnology, carbon nanotubes established materials have unlocked the latest routes for the upcoming novel functional materials. In short, we can say that the nanotubes made up of carbon especially have been used as new and utmost type of material applied for electronic and thermal applications. Although variety of nanotubes are available and being investigated by researchers with equal importance to

Introduction: Functionalized Carbon Nanotubes  15 harvest them at best. Some routes have been developed to handle and to manipulate the structure and properties of carbon nanotubes at an easier level. But much research is in need in the application of supercapacitors in this field. Some peoples recently, reporting carbon nanotubes-based supercapacitors with best performance. Literally on adding carbon nanotube with specific macromolecules that may offers enhanced conductivity of the produced material is the point of interest for nowadays researchers. Especially the best electrochemical property and environment friendly nature are two important aspects to think for nowadays. Looking forward to see these functional materials in 2D or 3D forms are also making a quite big area for the utmost development.

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18  Functionalized CNT for Biomedical Applications 48. Hu, H., Zhao, B., Hamon, M.A. et al., Sidewall functionalization of ­single-walled carbon nanotubes by addition of dichlorocarbene. J. Am. Chem. Soc, 125, 14893–14900, 2003. 49. Unger, E., Graham, A., Kreupl, F., Liebau, M., Hoenlein, W., Electrochemical functionalization of multi-walled carbon nanotubes for solvation and purification. Curr. Appl. Phys., 2, 107–111, 2002. 50. Kim, K.S., Bae, D.J., Kim, J.R., Park, K.A., Lim, S.C., Kim, J.J., Choi, W.B., Park, C.Y., Lee, Y.H., Modification of electronic structures of a carbon nanotube by hydrogen functionalization. Adv. Mater., 14, 24, 1818–1821, 2002. 51. Tagmatarchis, N. and Prato, M.J., Functionalization of carbon nanotubes via 1, 3-dipolar cycloadditions. J. Mater. Chem., 14, 437–439, 2004. 52. Chen, J.; Hamon, M.A.; Hu, H.; Chen, Y.; Rao, A.M.; Eklund, P.C.; Haddon, R.C., 1998. Solution properties of single-walled carbon nanotubes. Science 282, (5386), 95–98. 53. Esumi, K., Ishigami, M., Nakajima, A., Sawada, K., Honda, H., Chemical treatment of carbon nanotubes. Carbon, 34, 279–281, 1996. 54. Liu, J., Rinzler, A.G., Dai, H., Hafner, J.H., Bradley, R.K., Boul, P.J. et al., Fullerene pipes. Science, 280, 1253–1256, 1998. 55. Yu, R., Chen, L., Liu, Q., Lin, J., Tan, K.-L., Ng, S.C., Chan, H.S.O., Xu, G.-Q., Hor, T.S.A., Platinum deposition on carbon nanotubes via chemical modification. Chem. Mater., 10, 718–722, 1998. 56. Sham, M.-L. and Kim, J.-K., Surface functionalities of multi-wall carbon nanotubes after UV/Ozone and TETA treatments. Carbon, 44, 4, 768–777, 2006. 57. Wang, S.C., Chang, K.S., Yuan, C.J., Enhancement of electrochemical properties of screen-printed carbon electrodes by oxygen plasma treatment. Electrochim. Acta, 54, 21, 4937–4943, 2009. 58. Ma, P.C., Kim, J.K., Tang, B.Z., Functionalization of carbon nanotubes using a silane coupling agent. Carbon, 44, 3232–3238, 2006. 59. Sano, M., Kamino, A., Okamura, J., Shinkai, S., Self-organization of PEOgraft-single-walled carbon nanotubes in solutions and langmuir–blodgett films. Langmuir, 17, 17, 5125–5128, 2001. 60. Kong, H., Gao, C., Yan, D., Controlled functionalization of multiwalled carbon nanotubes by in situ atom transfer radical polymerization. J. Am. Chem. Soc., 126, 412–413, 2003. 61. Hamon, M.A., Hui, H., Bhowmik, P., Ester-functionalized soluble s­ ingle-walled carbon nanotubes. Appl. Phys. A, 74, 333–338, 2002. 62. Coleman, J.N., Khan, U., Gunko, Y.K., Mechanical reinforcement of polymers using carbon nanotubes. Adv. Mater., 18, 689–706, 2006. 63. Hu, L., Zhao, Y.-L., Ryu, K., Zhou, C., Stoddart, J.F., Gruner, G., Lightinduced charge transfer in pyrene/CdSe-SWNT hybrids. Adv. Mater., 20, 939–946, 2008. 64. Hecht, D.S., Ramirez, R.J.A., Briman, M., Artukovic, E., Chichak, K.S., Stoddart, J.F., Gruner, G., Bioinspired detection of light using a

Introduction: Functionalized Carbon Nanotubes  19 porphyrin-sensitized single wall nanotube field effect transistor. Nano Lett., 6, 2031–2036, 2006. 65. Star, A., Stoddart, J.F., Steuerman, D., Diehl, M., Boukai, A., Wong, E.W., Yang, X., Chung, S.-W., Choi, H., Heath, J.R., Preparation and properties of polymer-wrapped single-walled carbon nanotubes. Angew. Chem. Int. Ed., 40, 1721–1725, 20012001. 66. Steuerman, D.W., Star, A., Narizaano, R., Choi, H., Ries, R.S., Nicolini, C., Stoddart, J.F., Heath, J.R., Interactions between conjugated polymers and single walled carbon nanotubes. J. Phys. Chem. B, 106, 3124–3130, 2002. 67. Star, A., Liu, Y., Grant, K., Ridvan, L., Stoddart, J.F., Steuerman, D.W., Diehl, M.R., Boukai, A., Heath, J.R., Noncovalent side-wall functionalization of single walled carbon nanotubes. Macromolecules, 36, 553–560, 2003. 68. Star, A. and Stoddart, J.F., Dispersion and solubilization of single-walled carbon nanotubes with a hyperbranched polymer. Macromolecules, 35, 7516– 7520, 2002. 69. Cheng, F. and Adronov, A., Noncovalent functionalization and solubilization of carbon nanotubes by using a conjugated Zn-porphyrin polymer. Chem. Eur. J., 12, 5053–5059, 2006. 70. Yi, W., Malkovskiy, A., Chu, Q., Sokolov, A.P., Colon, M.L., Meador, M., Pang, Y., Wrapping of single-walled carbon nanotubes by a π-conjugated polymer: The role of polymer conformation-controlled size selectivity. J. Phys. Chem. B, 112, 12263–12269, 2008. 71. Kim, S., Hwang, J., Seo, J., Us, S., Production of CNT-taxol-embedded PCL microspheres using an ammonium-based room temperature ionic liquid: As a sustained drug delivery system. J. Colloid Interface Sci., 442, 147–153, 2015. 72. Mukherjeea, S., Kundub, B., Chandac, A., Sen, S., Effect of functionalisation of CNT in the preparation of HAp–CNT biocomposites. Ceram. Int., 41, 3766–3774, 2015. 73. Scapin, G., Salice, P., Tescari, S., Menna, E., De Filippis, V., Filippini, F., Enhanced neuronal cell differentiation combining biomimetic peptides and a carbon nanotube-polymer scaffold, 2014. https://pubmed.ncbi.nlm.nih. gov/25546847/ 74. Thakare, V.S., Das, M., Jain, A.K., Patil, S., Jain, S., Carbon nanotubes in cancer theragnosis. Nanomedicine (Lond.), 5, 1277–1301, 2010. 75. Bianco, A., Carbon nanotubes for the delivery of therapeutic molecules. Expert Opin. Drug Deliv., 1, 57–65, 2004. 76. Mehra, N.K., Jain, K., Jain, N.K., Pharmaceutical and biomedical applications of surface engineered carbon nanotubes. Drug Discovery Today, 1–10, 2015. http://dx.doi.org/10.1016/j.drudis.2015.01.006. 77. Vinoth, V., Wu, J.J., Asiri, A.M., Anandan, S., Simultaneous detection of dopamine and ascorbic acid using silicate network interlinked gold nanoparticles and multi-walled carbon nanotubes. Sens. Actuators B Chem., 210, 731–741, Published online 2020 Dec 16.

20  Functionalized CNT for Biomedical Applications 78. Lim, Y.T., Kim, S., Nakayama, A., Stott, N.E., Bawendi, M.G., Frangioni, J.V., Selection of quantum dot wavelengths for biomedical assays and imaging. Mol. Imaging, 2, 50–64, 2003.

2 Functionalized Carbon Nanotubes: Synthesis and Characterization Neelam Sharma1, Shubhra Pareek2, Rahul Shrivastava1 and Debasis Behera3* Department of Chemistry, Manipal University Jaipur, Jaipur (Rajasthan), India Department of Materials Engineering, Indian Institute of Science, Bangalore, India 3 School of Chemistry, Gangadhar Meher University, Amruta Vihar, Sambalpur, Odisha, India 1

2

Abstract

Numerous nanomaterials have been discovered and applied with the advancement of nanotechnology. Carbon nanotubes (CNTs) are one of them due to their amazing structure and magnificent properties, such as excellent conductivity, effective electrochemical and thermal stability, and great specific surface area. Owing to such features, CNTs have a wide series of medical, environmental, and industrial applications. In this chapter, the different routes of CNT synthesis including chemical vapor deposition (CVD), arc discharge, and laser ablation/Vaporization methods have been critically analyzed for the delivery of the desired application. The selectivity in the application can be modulated with tailored surface functionalization of CNT, which is further divided broadly into three categories. (i) through covalent bonds with a π-conjugated network of CNTs; (ii) through non-covalent bonds, π–π interactions, or ionic bonds, and hydrogen bonds for the attachment of different chemical groups; (iii) Endohedral filling functionalization (Inline filling) of hollow tubes of CNTs. These functionalizations give a deep insight into oxidation and activation, heteroatom doping, sulfonation, halogenation, catalytic chemical vapor deposition (CCVD), non-covalent functionalization, and nano-particle attachment. The presence of functional groups (surface oxides, principally hydroxyl, carboxyl, and carbonyl,) affects the modification of chemical reactivity, solubility, and other physical characteristics of CNTs. Numerous characterization techniques have been employed for the structural elucidation of CNTs such as FT-IR, UV–Vis, Raman, XRD, XPS, HR-TEM, FESEM, SAED, TGA, NMR. VSM, zeta potential DLS, and pH drift. *Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (21–48) © 2023 Scrivener Publishing LLC

21

22  Functionalized CNT for Biomedical Applications Keywords:  Nanotechnology, carbon nanotubes, synthesis, functionalization, characterization

2.1 Introduction In recent decades, Nanotechnology has been extensively studied, and several novel materials with various nanostructures (shapes and sizes) have emerged [1]. These nano-materials have several unique features which are not found in their micro-scale equivalents, offering up new possibilities for applications [2]. In the periodic table, one of the most adaptable elements is carbon because it can form a variety of C– C bonds (sp, sp2, and sp3-­hybridized bonds) [3]. In recent years, the synthetic allotropes of the carbon family have developed with the discovery of nano-onions [4], carbon nanohorns [5], carbon nanotubes (CNTs) [6], graphene [7], and other synthetic allotropes of carbon. CNTs have made significant contributions to chemistry, physics, material sciences, mathematical modeling, and other fields since their discovery [8]. Carbon nanotubes (CNTs) are the most promising and extensively investigated of all the carbon allotropes. CNTs have the easiest chemical composition and atomic bonding arrangement, but their structures and structural property relationships are extremely diverse and rich [9]. These were discovered in the soot of arc discharge equipment shortly after SumioIijima of the NEC Corporation synthesized fullerene in the lab in 1991 [10]. After two years, in 1993, two companies, IBM [11], and NEC [12] disclosed CNT with a single graphene layer. CNTs are the most widely studied allotrope of carbon because it has a unique hollow structure that gives it extraordinary electrical, thermal, and mechanical properties, such as high tensile strength better than any metal. The low density, high thermal conductivity exceeding diamond, high ductility, high electrical conductivity, high chemical, and thermal stability make it suitable for a wide range of applications [13]. CNTs are one of the most innovative structures available with a high aspect ratio (* 1000), and wide surface area. All these properties are dependent on their morphology, structure, and functions [14]. In the field of hydrogen storage [15] and generation [16], supercapacitors [17], biosensors [18], fuel cells [19], lithium-ion batteries [20], molecular sieves, electrochemical sensor [21] tissue engineering [22], and removal of pollutants [23] properties, CNTs exhibit potential applications. Various approaches have been developed to synthesize functionalized CNTs as well as to increase their solubility and dispersion in many solvents [24]. Due to their specific electronic, optical, magnetic, mechanical, and chemical properties that differ from those of their respective

Functionalized CNTs: Synthesis and Characterization  23

No. of citation

2500 2000 1500 1000 500 0

2017

2018 2020 2019 Year (2017-2021)

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Figure 2.1  Citation trends of published articles on functionalized carbon nanotubes.

bulk materials as well as constituent molecules or atoms, nanoparticles of modest diameters (10–20 nm) have garnered a lot of attention [25]. CNTs materials decorated with metal or metal oxide nanoparticles have gained significant interest in recent years [26]. In the past few decades, functionalized CNTs have generated widespread interest in commercial applications (Figure 2.1). The right combination of properties of the functionalized group makes CNTs suitable for use in many applications like sensing, biomedical, drugs delivery, energy storage, etc. Many of which have been patented. Some patents are discussed below. Scheinberg et al. have synthesized functionalized carbon nanotubes and filed a patent on May 22, 2018. In which they have explored the various application of carbon nanotubes on tumors [27]. Smith and Ghosn et al. have synthesized RGD-SWCNTs were used to deliver therapeutic agents for tumors and filed a patent on March 20, 2014 [28]. Myung et al. have synthesized metal and metal oxide co-functionalized SWCNTs and filed a patent in April 5, 2016, for the high performance of gas sensors [29]. Boday et al. have synthesized functionalized single-wall carbon nanotubes (FSWNTs) that were used in a printed wiring board (PWB)and filed a patent on September 5, 2017 [30]. Kobilka et al. discussed functionalized carbon nanotubes and filed a patent on March 19, 2019 [31]. Said et al. have synthesized CNTs functionalized with COOH (carboxyl) groups that are used for the solar adsorption refrigeration and filed a patent on November 1, 2016 [32]. To form macroscopic aggregates, Arnett et al. have synthesized DNA – functionalized SWCNTs through phosphodiester bonding and filed a patent on Jan. 7, 2020 [33]. This chapter aims to summarize (a) synthetic methods of CNTs. (b) Characterization Techniques, and (c) the systematic procedure to get

24  Functionalized CNT for Biomedical Applications the desired functionalization on CNTs. A classical balance has been presented between topics to increase readership among researchers, academicians, and students.

2.2 Synthesis Methods Figure 2.2 is a representation of the broad classification methods employed for the synthesis of the CNTs.

2.2.1 Arc Discharge CNTs were first produced by arc discharge between graphite electrodes [34]. Establishing a DC (direct current) between a pair of graphite electrodes under an inert atmosphere (argon or helium) at roughly 500 torr is required for this technique [35]. MWCNTs (Figure 2.3 (a)) are made by arc discharge without the use of a metal catalyst, whereas SWNCTs (Figure 2.3 (b)) require the use of mixed metal catalysts such as Co, Fe, and Ni [36]. CNTs synthesized via arc discharge method typically have a high degree of structural perfection [37], but a variety of variables (catalyst composition chamber temperature, and hydrogen present, concentration, etc.) Influence their structure and size [38]. Other electrodes [39] and compounds [40–42] have recently been used in the arc discharge synthesis

Methods of CNT synthesis

Chemical Vapor deposition

Arc Discharge Laser Ablation

Fixed bed reactors

Fluidized bed reactors

Microwave discharge

Laser-assisted

Hot filament

Aerosol-assisted

DC-glow discharges

Plasma-assisted

RF capacitive coupled plasmas

Floating catalyst

RF inductively coupled plasmas

Figure 2.2  The broad classification of methods employed for the synthesis of CNTs. Reprinted with permission [30] from Elsevier.

Functionalized CNTs: Synthesis and Characterization  25 (a)

(b)

Figure 2.3  Structure of (a) single-walled carbon nanotube (SWCNT), and (b) multiwalled carbon nanotube (MWCNT).

of CNTs. Using the arc discharge approach, Belgacem et al. [40] were able to synthesize MWCNTs doped with boron and nitrogen. Other research groups [41] have used arc discharge in the open air to make SWCNTSWCNT hybrids at a lower cost.

2.2.2 Laser Ablation Guo and coworkers were the first to disclose the synthesis of CNTs utilizing laser ablation in 1995 [43]. The laser ablation technology works on the same concept as arc discharge [44]. A solid graphite target mounted in a quartz tube is vaporized by a laser beam pulse at high temperatures (800 to1500 °C) in the presence of an inert atmosphere in this method [45]. The carbon-based soot is collected from the inside of the apparatus after the target has been vaporized [46]. Because of the high temperatures used in the synthesis, the CNTs produced by this approach have a high degree of structural perfection [47]. Furthermore, SWCNTs can be made without the use of MWCNTs. Compared to the arc discharge approach, the main disadvantages of this method include the high-power consumption with the lesser numbers of CNTs production [48]. The factors that influence the volume and quality of CNTs produced by laser ablation encompasses, (i) the chemical composition of the target material, (ii) the power of the laser and wavelength, and (iii) the distance between the substrates and the target [49]. Laser ablation allows high-purity CNTs to have a specified chirality (10,10) structure. In comparison to the chemical vapor deposition (CVD) method, the arc discharge and laser ablation procedures offer the benefit of producing higher-quality CNTs. The regular replacement of graphite electrodes and targets, the need for high vacuum conditions, and temperature restrain its ability to scale up CNT manufacture economically via arc discharge and laser ablation technologies [44].

26  Functionalized CNT for Biomedical Applications

2.2.3 Chemical Vapor Deposition Due to its simplicity, ambient pressure, low cost, and low temperature, chemical vapor deposition is the most used approach for the manufacture of carbon nanotubes [50]. The catalytic vapor phase deposition of carbon using carbon monoxide–hydrogen mixtures over Fe was first described in 1959 [51]. This procedure was used to make CNT from acetylene over Fe particles at 700°C in 1993 [52]. The CVD was first proposed as a method for largescale CNT production in 1996 [53]. Currently, the CVD is the technology for mass synthesis of various types of CNTs that have received the most attention [54]. To accelerate the formation of nanotubes, a gaseous hydrocarbon source (ethanol, acetylene, propylene, methane, ethylene, CO, etc.) is heated to 600-1000 °C with a transition metal catalyst such as Fe, Ni, Co, etc. (Figure 2.4) [55]. The CVD enables production on large-scale, excellent alignment [56], and controlled to development of nanotube [57]. Controlling the diameter and number of shells is also possible [58]. The CNTs produced through the CVD method have a larger defect density (amorphous carbon) than that of laser ablation, and arc discharge methods [59]. These flaws harm the CNTs’ thermal and electrical properties, as well as their structural properties [60]. The CVD approach is currently being used to regulate the synthesis of ultra-long CNTs with flawless structure. CNTs with good thermal electrical, and mechanical, properties can be made with this defect-free structure. The reader is directed to Zhang et al. [61] for a detailed explanation of the controlled synthesis of ultra-long CNTs. The integrated thermal plasma reactor and the CVD process have been employed recently to fabricate CNTs at atmospheric pressure [62]. (a) Graphite cathode

(b) CNT Deposit

Graphite Anode

Furnace Cooled collector

Laser

Inert gas

Inert gas

Graphite target DC source

(c) Split Furnace

Hydrocarbon source (CnHm)

Gas outlet

Gas + Precursor (and or catalyst)

Deposited CNts

Inert gas (N2/Ar)

Figure 2.4  CNT preparation techniques (a) arc discharge, (b) laser ablation, and (c) CVD.

Functionalized CNTs: Synthesis and Characterization  27

2.3 Characterization 2.3.1 Raman Spectroscopy One of the most used approaches for identifying structural faults in CNTs is Raman spectroscopy [63]. Raman spectra of CNTs depict have peaks at 1350 cm-1 that correspond to the D band whereas the peak at 1580 cm-1 represents the G band [64]. The G band is related to sp2 hybridization with symmetric graphite hexagonal lattices, while The D band corresponds to non-crystalline carbon (sp3 diamond-like) [65]. In other situations, a shoulder at the G band nearby 1620 cm-1 (D́ band) can be seen (Figure 2.5), which is related to (a)

intensity

G

(b) intensity

D

G D’

intensity

(c)

1200

1500 Raman shift (cm–1)

1800

Figure 2.5  The first-order Raman spectrum of (a) symmetric hexagonal lattice, (b) defective sp3 hybridized carbon along with shoulder band D́ (c) highly disordered single-layer graphene deposited on a substrate. Reprinted with permission from [The Royal Society].

28  Functionalized CNT for Biomedical Applications sp3 hybridized carbon that lacks in-plane symmetry with graphene [64, 66]. The intensity of the D band is proportional to the quantity of faulty carbon, and the ratio of the D and G bands’ intensities (ID/IG) is commonly employed to measure structural disorder. An increase in ID/IG suggests a loss of aromaticity in CNT rings due to faults caused by the hybridization of C-C sp2 to C-C sp3 (vide infra, section 2.3.2 FTIR) [67]. Raman spectroscopy can also be used for the investigation of chirality and count CNT walls.

2.3.2 Fourier Transform Infrared Spectroscopy (FT-IR) The surface of CNTs is evaluated qualitatively using Fourier transform infrared spectroscopy (FT-IR). In this method, infrared radiation is

(b) (a)

(b)

CNT-H CNT-Ac CNT-DDA

Absorbance 1734

(c)

1650 1580 1470

2920 2850

1630

3445

Transmittance (a.u)

(a)

4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1)

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

Figure 2.6  FT-IR spectra of CNT-H, CNT-Ac (CNT-COOH), and CNT-DDA (CNTdodecylamine) (a) transmittance data, (b) absorbance data, Reprinted with permission from [Elsevier].

Functionalized CNTs: Synthesis and Characterization  29 passed through the sample, and the fraction of this incident radiation absorbed at specific energy is calculated. In the spectrum, the frequency of a certain functional group is represented by each absorption peak. A distinctive band of about 3445 and 1630 cm-1 can be seen in the infrared spectra of raw CNTs (Figure 2.6 (a)), which is associated with O-H and carbon-­carbon double bonds stretching vibrations of aromatic rings on folded CNT respectively [68]. Bands at 3800–3200 cm-1 (OH stretching) and 1700–1600 cm-1 (OH bending) have been detected in some situations. These bands are caused by water molecules being absorbed by atmospheric moisture or by a purification process [69]. The appearance of bands at 2920 and 2850 cm-1 (Figure 2.6 (a)) supplement the evidence to asymmetric and symmetric stretching vibrations of the methylene (CH2) group, which are usually assigned to defects in rings of CNTs (Figure 2.6 (b)) (vide supra, section 2.3.1 Raman spectroscopy) [70].

2.3.3 Thermogravimetric Analysis (TGA) The purity and thermal stability of CNTs are studied by using thermogravimetric analysis (TGA) [71]. Depending on the information needed, the analysis might be done in an oxidizing or inert atmosphere. Some important parameters are analyzed like the initiation temperature (when weight loss begins), the final temperature (when weight loss ceases), the amount of weight loss at the initiation temperature, and the residual weight exhibited at the final temperature [72]. The degree of graphitization of the CNTs walls is indicated by the initiation temperature. The initiation temperature of CNTs with faulty hexatomic rings is lower, whereas the initiation temperature of more crystalline CNTs is higher [73]. The ultimate temperature reveals the nanotubes’ thermal stability: a higher temperature means more graphitizing CNTs, which means more thermal stability. The weight loss at the initiation temperature (100–300°C) represents the amount of contaminants and moisture in the sample, whereas the weight loss around 300– 700°C represents the disintegration of CNT aromatic rings. The amount of contaminants, such as metal particles utilized as catalyst and oxide produced by the oxidation of these metal particles, is also indicated by the residual weight [72].

2.3.4 Scanning Electron Microscopy (SEM) The structure and morphology of CNTs are studied using scanning electron microscopy (SEM). FEG-SEM (SEM with a field emission gun) is used to examine CNTs bundle and structural change after functionalization

30  Functionalized CNT for Biomedical Applications (a)

(b)

000104

3KV

X50.0K

600nm

15.0KV 7.7nm x200k SE(U)

200nm

Figure 2.7  Comparison of (a) “as-prepared” MWCNTs (600 nm) and (b) oxidized MWCNTs (200 nm) by SEM. The quantities given in brackets indicate the lateral resolution applied. Reprinted with permission from [Elsevier].

treatments, graphitization, and purification, as well as alignment, dispersion, and homogeneity [75]. Specific features, such as tube diameter and length, CNT entanglement and curvature, and the presence of amorphous carbon or other non desired carbon forms, can be revealed by microscope examination with enhanced resolution. Stobinski et al. [74] have investigated MWCNTs (Figure 2.7 (a)) using SEM and compared them with oxidized MWCNTs (Figure 2.7 (a)) with a lateral resolution of 600 nm and 200 nm respectively. The surface morphology of the prepared MWCNTs and oxidized MWCNTs (Figure 2.7 (b)) reveals the bundled structure of nanorods.

2.3.5 Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) of CNTs enables higher magnification observation of structural properties in individual nanotubes. Metal catalysts-based nanoparticles trapped in nanotube cores, flaws in CNTs wall like opening tips, carbonaceous particles, and amorphous carbon, resulting from the non-vaporized graphitic rod can all be identified [76, 77]. Moreover, the removal of the amorphous carbon from nanotubes was also evident after the oxidation of MWCNTs (Figure 2.8). Other structural factors that can be seen with TEM include tube diameter and length, entanglement, number of walls, and curvature [78]. In addition, the bundled structure of CNT nanorods of diameters ranging from 10 to 40 nm (Figure 2.8(b)) has also been reported in previous literature [74]. Surface morphological investigation through SEM revealed no structural changes as a result of the heat treatment, whereas TEM images revealed

Functionalized CNTs: Synthesis and Characterization  31 (a)

(b)

200 nm

20 nm

Figure 2.8  TEM image of oxidized, bundled MWCNTs. Scale bars are for (a) 200 nm and (b) 20 nm. Reprinted with permission from [Elsevier].

that the metal particles trapped in the nanotubes’ cores had been removed [76]. The surface morphology of the prepared MWCNTs and oxidized MWCNTs reveals the bundled structure of nanorods of diameters ranging from 10 to 40 nm.

2.3.6 X-Ray Diffraction (XRD) The crystalline structure of CNTs can be determined using X-ray diffraction (XRD) [78]. The XRD patterns of CNTs show two primary diffraction peaks: (002) hexagonal graphite structure reflection at about 2θ = 26 degrees and (100) graphite base plane diffraction roughly at 2θ = 43 degrees [79]. The XRD technique is also used to investigate the structure

(002)

Intensity/a.u.

g-MWCNTs MWCNTs

(100)

(004)

(110)

(002) (100)

10

20

30

40 50 60 2θ/degree

70

80

90

Figure 2.9  XRD pattern of graphitized multi-walled carbon nanotubes (g-MWCNTs) and pristine MWCNTs. Reprinted with permission from [Elsevier].

32  Functionalized CNT for Biomedical Applications of CNTs changes following graphitization, functionalization, and purification. Using XRD patterns (Figure 2.9), Xue et al. [80] investigated the effect of graphitization on CNTs and found that after the treatment, the peak at (002) became sharp and narrow. Furthermore, the authors observed new peaks at 2θ = 77.5 degrees and 2θ = 54.3 degrees ascribed to (110) and (004) diffraction, respectively.

2.3.7 X-Ray Photoelectron Spectroscopy (XPS) The components on CNT surfaces are analyzed using X-ray photoelectron spectroscopy (XPS), which provides information of the functional groups linked to CNTs wall to a 10 nm depth [78]. The O1s and C1s peaks in the XPS survey spectra of raw CNTs have binding energies of 533 and 284 eV, respectively. The peak in O1s can be due to flaws, moisture, or O2 and CO2 absorbed on the CNTs from the environment. Other peaks (such as N1s at 400 eV) can be seen in the CNT spectrum depending on the post-processing and fabrication method [81]. Six peaks (Figure 1.5b) can be used to deconvolute the C1s peak: C-C sp 3 (~285.2 eV), C-C sp 2 (~284.6 eV), C = O (~288.9 eV), O-C = O (~289.8 eV), C-O (~286.8 eV), and π-π* shakeup feature (~ 291.0 eV) [82]. By graphitization, functionalization, or Purification, procedures show modification on the crystalline structure of CNTs, resulting in a change in peak area (Figure 2.10) [78, 80].

Intensity

g-MWCNTs

gMWCNTs MWCNTs 66.33% 67.15% C=C 24.39% C-OH 20.44% 5.22% 1.96% C=O 6.51% O=C-OH 8.02%

MWCNTs

282

284

Raw C=C C-OH C=O O=C-OH 286 288 290 Binding Energy/eV

292

294

Figure 2.10  XPS spectra of C1s for graphitized multi-walled carbon nanotubes (g-MWCNTs) and pristine MWCNTs. Reprinted with permission from [Elsevier].

Functionalized CNTs: Synthesis and Characterization  33

2.4 Functionalized Routes of CNTs 2.4.1 Surface Oxidation Controlled oxidation is commonly used to introduce oxygen-containing groups like phenol, ketone, carboxyl group, lactone, acid anhydride, ether, and so on to CNT surfaces [83]. In polar solvents such as water, these oxygen-­containing groups can improve the wettability or hydrophilicity of the CNT surface. These oxygen-containing groups, on the other hand, can act as anchoring sites for further functionalization via covalent, electrostatic, or hydrogen bonding. The strength of oxidants and the oxidation circumstances dictate the extent of oxidation. Hydrogen peroxide, Nitric acid permanganate, persulfate hypochlorite, chlorates, hypochlorite, dichromate, ozone, nitric oxide, and oxygen are all instances of oxidants. Aqueous nitric acid, either concentrated or diluted, is the most commonly used oxidant for carbon materials [84]. This method is also useful for CNT purification because it removes metallic impurities and amorphous carbon [85]. Nitric acid is controllable and an efficient oxidant for the formation of surface groups by controlling acid concentration, oxidation, and temperature.

2.4.2 Doping Heteroatoms During the one-step CNT synthesis, an effective method for doping heteroatoms like boron and nitrogen on CNTs is frequently achieved. In the case of N-doped nanotube synthesis, the carbon and nitrogen sources are a combination of ammonia [86] and hydrocarbon and or organic amine [87] and hydrocarbon. These reactants were slowly injected with argon into a heated quartz tube furnace holding quartz or ceramic boat carrying an iron-rich catalyst [88]. Organic borane is utilized instead of organic amine in the synthesis of B-doped CNTs, which uses the same CVD technique as the production of N-doped CNTs. Doping heteroatoms is an effective way of fine-tuning the structural and electrical features of nanotubes.

2.4.3 Alkali Activation To modify the texture of carbonaceous materials, activation with alkali or CO2 is utilized, which increases the specific surface area and pore volume [89]. This approach is also used to activate carbon nanotubes. The mass ratio of KOH to CNT was established at 4:1 before activating at 600°C, 700°C, or 800°C for 1 hour under nitrogen flow. Before filtering, the activated CNTs

34  Functionalized CNT for Biomedical Applications were washed with a weak HCl aqueous solution to eliminate alkali species, followed by drying at 100°C. The as-prepared activated CNTs have a higher specific surface area and pore volume, as well as improved hydrogen storage and migration properties [90]. Furthermore, the conductivity and electrochemical performance of alkali-activated CNTs as electrode materials for supercapacitors are shown to be superior to that of pristine CNTs [91].

2.4.4 Sulfonation Environmentally acceptable solid–acid catalysts for esterification, rearrangement, dehydration and condensation processes have been produced using carbon compounds functionalized with sulfonic acid [92]. Sulfonation can also be accomplished by treating CNTs with ammonia sulfate. Before heating at 235 °C for 30 minutes, ammonia sulphate solution was stirred with a sufficient number of CNTs. SO3 is formed when ammonia sulphate decomposes, forming a surface with –SO3H groups [93].

2.4.5 Halogenation To increase the hydrophobicity of carbon surfaces, surface fluorination was explored. Several approaches were used to functionalize fluorine onto carbon surfaces. Fluorinated CNTs were directly produced for 12 hours at 350  °C in a fluorine gas environment utilizing an acceptable number of CNTs [94]. Fluorination made the exterior of the CNTs hydrophobic, allowing them to be disseminated in an epoxy matrix bromination of the CNTs is an effective approach for creating anchoring groups on the CNTs surfaces for subsequent replacement of a variety of nucleophiles such as anilines, alcohols, thiols, amines, and, among others. Bromine halogenation is accomplished by directly treating the CNTs with bromine at high temperatures over Lewis acid catalysts [95].

2.4.6 Grafting The post grafting technique may be used to impart a wide range of surface functional groups onto CNT surfaces (Figure 2.11). The CNT surface already contains specific surface functional groups in several of these approaches, which may be further substituted by other organic groups. The pre-existing functionalities are generally produced by oxidizing CNTs with sulfuric acid/nitric acid during boiling. In some cases, CNT surfaces can also be directly coupled with functional groups.

Functionalized CNTs: Synthesis and Characterization  35 O S

O

O

O

S MgClS

S

CH2 Si OH

HO

NH2

CH2 CH2 CH2 O Si O O

CH2

N H

S

NH2

CH2

SH

S O

NH2

SH

N H

OH

CH2 CH2 CH2 Si O O

SH

H2N

O

COOH C CH NH n NH (CH2)4CH NH

SO

Cl

Cl2

O (CH2)4NH2

F

Br

Or

F

Br

N N

2H 5)3 O

OH

HNO3 or H2O2 or (NH4) 2S2O4

tive

OH

or a + n niline itril e deriva

CF3

8 r C3 F CF4 o a Plasm r 2 B r R o F2

H2 N(C ex H2 cess )3S i(O C

O

O

CF3

H2N(CH2)3Si(OC2H5)3

Cl

L-lysine

NH2

Cl

O N

R' OR

R

R

O O

O

N

NH2

NH2

OR

R'

Figure 2.11  Grafting reactions to functionalized CNTs surface via covalent bonds.

2.4.6.1 Grafting via Oxygen-Containing Groups On oxidant treated CNTs surface, carboxyl groups react with thionyl chloride to form acyl chloride groups, which work as an intermediate for various organic reactions. Cysteamine can react with acyl group by amidation reaction and graft thiol group. The presence of thiol group in CNT helps in attaching metal nanoparticles on the side of CNTs, which serve as catalysts and sensors [96]. The other use of acyl group can be grafting of chitosan on the surface of CNT to produce a new CNT/chitosan composite with application in the field of bone tissue engineering. Initially, 0.02 g of K2S2O8 is mixed with 0.5 g of powdered chitosan for the duration of 20 h then lactic acid (0.055) is added gradually up to 2 h. Further, the reaction

36  Functionalized CNT for Biomedical Applications is stopped by using THF (10 mL) followed by ice drying (freeze-drying) thereafter methanol was used to rinse the dried sample. Afterward, the prepared sample is reacted with an acyl derivative of CNTs in the presence of CH3COOH for 24 h at 75°C in vigorous stirring. Furthermore, the use of acetic acid eliminates the unreacted part of chitosan [97]. For methyl orange degradation reaction, L-lysine and acyl chloride react with each other through an amidation reaction to form poly-lysin embedded CNTs. It has been shown to have improved wettability when it comes to depositing and dispersing titanium dioxide nanoparticles. For the methyl orange degradation process, the resultant composite provided increased photocatalytic activity. Metal-organic salts may react with acyl derivatives of CNT to create CNT polymers that can be used in reversible addition-fragmentation chain-transfer polymerization in addition to the amidation process. CNT chain transfer agents are formed when magnesium chloride di-thiopropanoate reacts with acylchloride groups on CNT surfaces [98]. A variety of groups may be grafted using the esterification depending on the carboxyl groups on CNT surfaces. For example, imidazolium functionalized carbon nanotubes have been produced, and the resulting nanohybrid composites with organometallic complexes show efficient and effective heterogeneous, which works as a  hydrogen transfer catalyst for the reduction of cyclohexanone in the presence of 2-propanol as the hydrogen source [99]. The 3-aminopropyltriethoxysilane functionalized CNTs were also found to have promising CO2 absorption capabilities and were chosen as CO2 adsorbents for cyclic CO2 capture study [100]. At 80°C, oxidized CNTs dispersed in water were combined with 3-aminopropyltriethoxysilane dissolved in acetone for 30 minutes under vigorous stirring. Filtration with distilled water and acetone separated the silane-functionalized CNTs before drying overnight [101].

2.4.6.2 Grafting via Diazonium Compounds Diazonium compound has been used to graft aryl or aryl derivatives directly onto CNTs surface. It is a flexible grafting method that employs diazonium compounds. The interaction between diazonium salts and CNTs in a typical aqueous solution-based grafting procedure is a free radical chain reaction begun mostly by diazo-anhydride homolytic decomposition and partially by direct oxidation of CNTs by diazonium compounds. Once generated, aryl radicals can be covalently linked to carbon nanotubes (CNTs).

Functionalized CNTs: Synthesis and Characterization  37

2.4.6.3 Other Grafting Methods Although numerous grafting methodologies for CNT surface functionalization have been investigated, there are still certain grafting methods that are often used on carbon surfaces and can be used to functionalize CNT surfaces. A one-step procedure is used to directly functionalize carbon compounds. Under microwave irradiation, activated carbon was combined with amines, alcohols, or thiols for a solvent-free reaction, and the organic groups were directly connected to the double bonds. The microwave-assisted (MA) reactions were carried out in a controlled environment with temperatures below 200°C and duration of fewer than 100 minutes [102]. The cycloaddition process between cyclic nitrones and CNTs under sonication can covalently link cyclic nitrones to CNT surfaces, as demonstrated in Figure 2.10. By reacting with aniline precursors, nitrite, and acid, carbon black was directly functionalized with nitrophenyl, phenyl, and phenyl-azo-aniline [103].

2.4.7 Non-Covalent Functionalization of CNTs Non-covalent functionalization approaches based on the ‘p–p’ interactions between CNTs and guest molecules have been used to preserve the sp2 CNT structures and consequently their electrical properties. It is found that p–p stacking was used to functionalize the CNT sidewall with protein. Using the robust p–p interaction involving CNTs and the pyrenyl group, the CNTs were first designed and synthesized with succinimidyl ester and 1-pyrenebutanoic acid. Further, the amide bond is formed through the nucleophilic substitution of N-hydroxy succinimide on the protein [104].

2.4.8 Deposition on Functionalized CNTs Functionalization may be described as the insertion of nanoparticles on carbon nanotubes. The addition of nanoparticles allows CNT applications to be expanded and introduces additional properties such as catalytic and electrochemical activity. Precursors attached to CNTs in the presence of surface functional groups for attaching metal ions or metal nanoparticles generate mixed metal oxides and metal oxides [105]. The most widely used functional groups on CNTs are carbonyl and carboxyl groups. This approach is also known as impregnation, and it is a simple and affordable technique. Nano-particles generated on flawless CNT surfaces by the impregnation method rely solely on ‘particle–support physical contact’, which can effortlessly result in leaking and degradation of

38  Functionalized CNT for Biomedical Applications the nano-particles due to the lack of surface functional groups that serves as an anchoring site. Taking MnO2 as an example, the CNT was oxidized employing nitric acid and functionalized with oxygen-containing groups. Separately, 0.5 g of functionalized CNTs dispersed in 50 mL of toluene and a determined quantity of manganese (II) acetylacetonate dissolved in 50 mL of toluene were refluxed at 110°C for 5 h with a nitrogen flow before the two slurries were blended in a bigger flask and refluxed at 110°C overnight. The slurry was kept at room temperature for cooling, which is further filtered, washed, and desiccated before being annealed at 400°C for 6 hours in a nitrogen environment [106]. In the aerobic oxidation process of benzyl alcohol, these manganese oxides functionalized CNTs are used as catalytic substrates for palladium catalysts. Mixed metal oxides of cerium and copper were placed on carbon nanotubes pretreated with HNO3 [107]. Impregnation was used to create highly distributed RuO2 nanoparticles on CNTs.

2.4.9 Physiochemical Approaches Nanoparticle-deposited CNTs may be made using a variety of physical processes. Sputtering deposition, evaporation deposition, electron, and ion-beam irradiation deposition are some of the physical processes used. The shape, size, dispersion, and homogeneity of the nanoparticles may be controlled using these strategies.

2.4.10 Electrochemical Deposition Electrochemical deposition permits the formation of metal Nps attached on CNTs by the reduction of the metal complexes i.e., platinic chloride acid, ammonium platinic chloride, and auric chloride acid, by electrons on cathodes [105]. CNTs function as conducting wires and work as a support for metal Nps deposition while exhibiting no reactivity with the metal precursors. Altering the parameters such as nucleation potential, the concentration of metal precursors, and process time indorses a difference in the homogeneity and size of metal Nps on the surfaces of CNTs. The electrochemical deposition has the benefit of producing exceptionally pure metal NPs functionalized on carbon nanotubes with robust metal support contacts in a very short process time [108]. The downside of the electrochemical deposition process, is the creation of large metal NPs on CNTs surface, typically ranging from 10 to 100 nm in size [109]. It has been reported that nickel nanoparticles can be generated on carbon nanotubes. Nickel particles were dispersed more uniformly on the surface of functionalized CNTs

Functionalized CNTs: Synthesis and Characterization  39 than on the surface of pristine CNTs after electrochemical deposition on both functionalized and pristine CNTs [110]. The shape of nickel oxide on carbon nanotubes is affected by variations in deposition time and electric current [111].

2.4.11 Electroless Deposition Chemical reduction in the existence of reducing chemicals is required for electroless deposition of nanoparticles on CNTs. During the electroless deposition process, direct reduction occurs between the CNTs and metal precursors [105]. Nevertheless, the use of electroless deposition is limited by the fact that metal ions must have greater redox potentials than CNTs in order to be reduced to nanoparticles on CNTs support [112]. This limitation has been solved by using a ‘substrate-enhanced electroless deposition’ process that includes supporting CNTs with a metal substrate that has a lower redox potential than the targeted metal precursors [98]. A Pt/Mo/ Ru/graphene–CNT catalyst was created by chemically reducing metal precursors at room temperature using sodium borohydride (NaBH4) as the reducing agent [113].

2.5 Conclusion This chapter gives an overview of current advances in the synthesis, characterization, and functionalization of carbon nanotubes. They are mostly employed in electronic gadgets, polymer nanocomposites, and medicinal materials for biomedical purposes as reinforcement. CNT uses are projected to grow as the understanding of synthesis methods improves, as well as future breakthroughs in surface modification. With changes in interfacial, sorption, electrical, and acid-base characteristics, heteroatom-doped CNTs can give more active sites in the meantime. Sulfonation transforms CNTs into solid acids, while diazonium functionalization allows aryl compounds to be covalently grafted onto CNT surfaces. CNTs and functionalized CNTs have been extensively used and studied in a variety of reactions, including CO/H2 conversion to hydrocarbons or alcohols, hydrogenation, oxidation, dehydration, C–C coupling reactions, amino synthesis, and breakdown, photocatalytic reactions, CNT synthesis, and so on. With the promising inherent features of CNTs and functionalized CNTs, there are still many applications in catalysis to be investigated in depth. In the future, we may see more designed functionalized CNTs supported catalysts with higher performance in eco-friendly processes, like

40  Functionalized CNT for Biomedical Applications plastic degradation for cellulose converted into ethanol, fuel, air pollution treatment, wastewater treatment, renewable energy conversion, and so on, to solve energy and environmental problems, which are still difficult to solve today.

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Functionalized CNTs: Synthesis and Characterization  45 77. Goornavar, V., Jeffers, R., Biradar, S., Ramesh, G.T., Utilization of highly purified single wall carbon nanotubes dispersed in polymer thin films for an improved performance of an electrochemical glucose sensor. Mater. Sci. Eng. C, 40, 299, 2014. 78. Chong, C.T., Tan, W.H., Lee, S.L., Chong, W.W.F., Lam, S.S., Medina, A.V., Morphology and growth of carbon nanotubes catalytically synthesised by premixed hydrocarbon-rich flames catalyst-supported. Mater. Chem., 197, 246, 2017. 79. Silambarasan, D., Surya, V.J., Iyakutti, K., Asokan, K., Vasu, V., Kawazoe, Y., Gamma (γ)-ray irradiated multi-walled carbon nanotubes (MWCNTs) for hydrogen storage. Appl. Surf. Sci., 418, 49-55, 2016. 80. Xue, Y., Zheng, S., Sun, Z., Zhang, Y., Jin, W., Alkaline electrochemical advanced oxidation process for chromium oxidation at graphitized multiwalled carbon nanotubes. Chemosphere, 183, 156, 2017. 81. Ferreira, F.V., Francisco, W., Menezes, B.R.C., Brito, F.S., Coutinho, A.S., Cividanes, L.S., Correlation of surface treatment, dispersion and mechanical properties of HDPE/CNT nanocomposites. Appl. Surf. Sci., 389, 921, 2016. 82. Ferreira, F.V., Franceschi, W., Menezes, B.R.C., Brito, F.S., Lozano, K., Coutinho, A.R., Dodecylamine functionalization of carbon nanotubes to improve dispersion, thermal and mechanical properties of polyethylene based nanocomposites. Appl. Surf. Sci., 410, 267, 2017. 83. Fanning, P.E. and Vannice, M.A., A DRIFTS study of the formation of surface groups on carbon by oxidation. Carbon, 31, 5, 721, 1993. 84. Klink, S., Ventosa, E., Xia, W., La Mantia, F., Muhler, M., Schuhmann, W., Tailoring of CNT surface oxygen groups by gas-phase oxidation and its implications for lithium ion batteries. Electrochem. Commun., 15, 1, 10, 2012. 85. Guo, Z., Chen, Y., Li, L., Wang, X., Haller, G.L., Yang, Y., Carbon ­nanotube-supported Pt-based bimetallic catalysts prepared by a microwaveassisted polyol reduction method and their catalytic applications in the selective hydrogenation. J. Catal., 276, 2, 314, 2010. 86. Chizari, K., Vena, A., Laurentius, L., Uttandaraman, S., The effect of temperature on the morphology and chemical surface properties of nitrogen-doped carbon nanotubes. Carbon, 68, 369, 2014. 87. Adjizian, J.J., Leghrib, R., Koos, A.A., Suarez-Martinez, I., Crossley, A., Wagner, P., Grobert, N., Llobet, E., Ewels, C.P., Boron-and nitrogen-doped multi-wall carbon nanotubes for gas detection. Carbon, 66, 662, 2014. 88. Dong, J., Qu, X., Wang, L., Zhao, C., Xu, J., Electrochemistry of ­nitrogen-doped Carbon Nanotubes (CNx) with different nitrogen content and its application in simultaneous determination of dihydroxybenzene isomers. Electroanalysis (N.Y.N.Y.), 20, 1981, 2008. 89. Romero-Anaya, A.J., Ouzzine, M., Lillo-Ródenas, M.A., Linares-Solano, A., Pherical carbons: Synthesis, characterization and activation processes. Carbon, 68, 296, 2014.

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3 Carbon Nanotubes: Types of Functionalization Manilal Murmu1,2, Debanjan Dey1,2, Naresh Chandra Murmu1,2 and Priyabrata Banerjee1,2* Surface Engineering and Tribology Group, CSIR-Central Mechanical Engineering Research Institute, Mahatma Gandhi Avenue, Durgapur, India 2 Academy of Scientific and Innovative Research (AcSIR), AcSIR Headquarters CSIR-HRDC Campus, Kamla Nehru Nagar, Ghaziabad, India

1

Abstract

The carbon nanotubes (CNTs) have emerged as novel functional materials with the progress of materials science and nanotechnology. The CNTs are sp2 hybridized rolled up graphene sheets having one-dimensional carbon possessing aspect ratio > 1000. The important class of CNTs like single walled carbon nanotubes (SWNTs), multi-walled carbon nanotubes (MWNTs) etc., has been briefly outlined. This chapter covers the most significant as well as efficient chemical or physical functionalization approaches for obtaining desirable properties of mostly SWNTs and MWNTs based on covalent and non-covalent interactions. The desired properties such as chemical, physical, mechanical, electrical, transportation properties, etc., of the CNTs can be achieved through its controlled functionalization. The mechanism of the functionalization of CNTs has been discussed briefly. The advantages and disadvantages of various chemical functionalization methods of CNTs have also been overviewed. The outlook of its future applications based on its functionalization has been predicted. Keywords:  Carbon nanotubes, chemical functionalization, covalent functionalization, non-covalent functionalization

*Corresponding author: [email protected]; Website: http://www.priyabratabanerjee.in Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (49–74) © 2023 Scrivener Publishing LLC

49

50  Functionalized CNT for Biomedical Applications

3.1 Introduction The carbon nanotubes (CNTs) are hollow tubular structured carbon allotropes. The discovery of CNTs was first revealed from high resolution current arc discharge products through high resolution electron microscopy by Iijima in 1991 [1]. Later in 1993, nanotubes with single layer named as single walled carbon nanotubes (SWNTs) were revealed by the same arc process with the catalytic particles [2, 3]. The hollow concentric cylindrical carbon tubules were named as multi-walled carbon nanotubes (MWNTs). In 1991, CNTs have emerged as novel functional materials with the progress of materials science and nanotechnology. The CNTs are synthesized using several methods such as (a) arc-­ discharge [4] (b) laser ablation [5], (c) chemical vapor deposition (CVD) [6], (d) catalytic gas-phase growth starting with carbon monoxide or other carbon sources (e.g., HiPco methods) [7]. The first two methods involve the vaporization of carbon in an inert atmosphere, whereas CVD and HiPco are based on catalytic decomposition of hydrocarbons. The synthesis of CNTs has advanced to the point that patterns of aligned MWNTs or SWNTs can be engineered. The applications of CNTs ranges from high performance nano-electronics and re-enforced composite to hydrogen storage, field emission devises, sensors and probes [8], conductive coating, super capacitor electrodes [9], in medical sciences such as drug, gene and peptide delivery, tissue regeneration, imaging and diagnostics [10]. In this chapter, the most significant strategies used for the functionalizations of CNTs have been described briefly. Various schemes for explaining different modifications of these carbon nanostructures have also been elucidated. Since, the CNTs have diverse applications, understanding of the insight and mechanism of its functionalizations in order to impart various desired properties is very necessary so that these CNTs might find promising applications in various fields.

3.2 Carbon Nanotubes The CNTs are hollow carbon structures with one or more concentric cylindrical walls. The CNTs are sp2 hybridized rolled up graphene sheets having one dimensional carbon form that can be open ended or capped, having high aspect ratio (>1000) with diameter as small as 1 nm and a length of several micro-meters [11]. CNTs are designated as single walled, SWNTs double-walled carbon nanotubes DWNTs, a few-walled carbon nanotubes

Carbon Nanotubes: Types of Functionalization  51 (FWNTs) or multi-walled, MWNTs depending on the number of walls present in it. The structure and properties of CNTs depends upon atomic arrangement through which the graphite sheets were rolled cylindrically to form the CNTs as shown in Figure 3.1 [12]. Broadly, the CNTs are categorized as SWNTs, DWNTs, FWNTs and MWNTs based on the atomic arrangements, its diameter, tube length and nanostructure made up of several concentric shells. The structure of CNTs as a function of number of sidewalls, SWNTs as function of chirality i.e., zigzag, chiral, and armchair are shown in Figure 3.1 (a), DWNTs in Figure 3.1 (b), and MWNTs in Figure 3.1(c). The SWNTs comprised of a cylindrically rolled single graphene sheet having 1 nm diameter and lengths in centimeters [2, 3]. The arrays of

(a)

chiral (m=n) / Zigzag (n,0) armchair (n,n)

ф

T (b)

a1 a 2 (c)

Figure 3.1  Structure of CNTs as a function of number of sidewalls (a) SWNTs as function of chirality (zigzag, chiral, and armchair), (b) DWNTs, and (c) MWNTs [Reproduced from Ref. [12]. Copyright © 2015 Tîlmaciu and Morris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)].

52  Functionalized CNT for Biomedical Applications cylindrical tubes in MWNTs are concentrically arranged with 0.35 nm separation distance like the basal planes in graphite [13]. The cylindrical tube diameters in MWNTs are within 2 - 100 nm and tens of microns lengths [14]. The graphene sheets are rolled along (m, n) lattice vector to form SWNTs as shown in Figure 3.1 (a). Diameters as well as chirality of SWNTs are determined by the (m, n) indices. Based on the (m, n) indices, there are three types of SWNTs. When, the values of n = m, it forms an “armchair” type (= 0º) nanotube. The SWNT formed is of “zigzag” type (= 30º) when m = 0. On the other hand, a “chiral” nanotube is formed if n ≠ m, and lies within 0º and 30º as shown in Figure 3.1 (a). Chirality of CNTs is the function of (m, n) value. It affects mechanical, optical and electronic properties of the CNTs. It has been reported that the metallic nature is exhibited by all armchair and one-third of SWNTs, while, remaining CNTs with typical diameters behaves like semiconductors [15, 16].

3.3 Functionalization of Carbon Nanotubes Curvature of graphene sheets facilitates variations of pyramidalization angle as well as it causes the misaligning of p-orbitals. These changes impart enhanced chemical reactivity of CNTs towards addition reactions. Furthermore, these changes make the sidewalls of CNTs more susceptible towards chemical reactivities compared to planar sheets of graphene.

3.3.1 Covalent Functionalization The covalent functionalizations of CNTs are accomplished through covalent bond formation by the functional groups and carbon atoms of CNTs. This functionalization is made at the terminal boundaries of CNTs or exterior walls [17]. The CNTs are chemically modified through (a) sidewall and (b) defect functionalizations. The carbon atoms of the side walls or graphene layers of CNTs are sp2 hybridized, as well as it possesses conjugated π-bonds. Hence, these sp2 hybridized carbon atoms can be transformed to sp3 system and conjugation of π-bonds can simultaneously be ceased. It can be accomplished by reacting CNTs with highly reactive chemical entities such as fluorine, etc., as shown schematically in Figure 3.2 (a). Similarly, the CNTs can also be modified chemically by forming defect(s) on the graphene layer as shown schematically in Figure 3.2(a). Both the sidewall and defect functionalization give rise to form covalent bonds between the active centers of reactive species with the sp2 hybridised carbon atoms of CNTs. Upon, initial functionalization of these CNTs, further

Carbon Nanotubes: Types of Functionalization  53 CI CI Carbene

ROOC N Nitrene EtOOC EtOOC

NH2 Flurination and derivative reactions

n Diels-Alder

Nucleophilic Cyclopropanation

R

F c d

RMgBr/RLi

a

T>450 °C Dehydrogenation

R

H

b R : Alkyl, Aryl, etc.

Radical (R•) attachment

Carboxylation Silanization

Amidation O

e

h

O

NH-R-NH2

Si-R

g

O Thiolation

f

O OR

O

OH

Esterif ication

i

SH-R

Polymer Grafting

Metal ions (Au, Ag etc.) Metal nanoparticles

(M1)n-(M2)m

Grafting from 2 Grafting to 1 Monomer 1

(M1)n Monomer 23

O O-(M-M)n

1 Caboxylic CNTs reacts with reactive polymers 2 Functionalized CNTs act as initiators to initiate polymerization 3 Living polymerization, CNT copolymer can be obtained; n, m; Degree of polymerization

Figure 3.2  Functionalization of CNTs using different approaches.

derivatives of the CNTs can be obtained with subsequent chemical modifications. The side wall of CNTs was modified using highly reactive fluorine at 325 °C (fluorination) as shown in Figure 3.2(a). It was also reported that this fluorination process is reversible in presence of anhydrous hydrazine [18]. It was reported that the carbon fluorine i.e., C-F bond formed

54  Functionalized CNT for Biomedical Applications in CNTs are weaker than that formed in the alkyl fluorides. Thus, the fluorine atoms of the C-F bonds in CNTs can be easily be substituted and furthermore additional derivatives of CNTs can be achieved [19, 20]. For instances, the alkylation of the fluorinated CNTs can be achieved by using Grignard reagent (RMgX) or alkyl lithium (RLi) as shown in Figure 3.2 (a), where R represents the alkyl or aryl group [21, 22]. Recently, several efficient approaches are executed for the functionalization of CNTs, for instances, Diels-Alder reaction, carbine, and nitrene addition like hydrogenation as shown in Figure 3.2 (b) [23], cycloaddition reactions as shown in Figure 3.2 (c) [24–26]. The alkylation or arylation of the CNTs may also be performed using radical attachment as shown in Figure 3.2 (d) [17]. Furthermore, bromination, chlorination [27], azomethine ylides [28], and so on are also adopted for functionalization of the side wall carbon atoms of the CNTs. Another method of CNTs modification through covalent bond formation approach is the defect functionalization [17]. The defect sites such as holes in the side walls, open endings, pentagonal or heptagonal anomalies may be present in hexagonal graphene structure in the CNTs. Furthermore, the oxygenated carbon centers are also considered as defects in the CNTs. The defects on the side walls or the open ends of CNTs are chemically formed through oxidative pathway using H2SO4, HNO3 and both used in combination [29]. Likewise, strong oxidants like KMnO4 [30], ozone [31, 32] and reactive plasma [33, 34] are also used for defects formation on CNTs through oxidative process. The bonding of carboxylic acid or hydroxyl unit with defects formed on the CNTs stabilized it chemically (vide Figure 3.2). Thus, these CNTs find their applications being starting materials for additional functionalization, for instances, amidation that is shown in Figure 3.2(e) [17], esterification as shown in Figure 3.2(f) [35], thiolation and/or subsequent incorporation of metal nanoparticles as shown in Figure 3.2 (g) [36], silanization as shown in Figure 3.2(h) [32], polymer grafting as shown in Figure 3.2(i) [37]. Furthermore, the alkylation or arylation [38], and biomolecule derivatives [39] can also be prepared from this functionalization technique. In such chemical modification the polar groups are being attached in the walls of CNTs. Thus, these sorts of chemical modification transform the hydrophobic CNTs towards hydrophilic one. Additionally, the strong bonds between the CNTs and the functional moieties in the polymers are formed at the interface which gives CNTs nanocomposites. These CNT based nanocomposites may exhibit extraordinary mechanical as well as chemical stability.

Carbon Nanotubes: Types of Functionalization  55 Furthermore, Diels-Alder reactions accompanied by redox polymerization has also been reported to be very efficient chemical modification technique for CNTs occurring at mild reaction environments, low temperature, stable in water and hydroxyl surface initiator. Herein, hydroxyl groups are incorporated CNTs sidewalls initially reacting it with furfuryl alcohol through Diels-Alder reaction. Subsequently, the furfuryl alcohol modified CNTs (CNTs-OH) was further modified by grafting the co-polymer which was started by Ce(IV)/HNO3 redox system utilizing biocompatible hydrophilic poly(ethyleneglycol) methyl ether methacrylate (PEGMA) and carboxyl-rich acrylic acid (AA) as monomers as shown in Figure 3.3. The advantage of the synthetic procedure is that the reaction was accomplished at ambient condition in absence of organic solvent without involving any expensive and toxic substances [40]. Furthermore, literature also reveals that the CNTs are also functionalized using ionic liquids in which the ionic liquids exhibit strong π-cation interactions with the CNTs and thereby synergistically enhances its various properties [41]. The ionic liquids are fluids at room temperature, this property make it capable to disperse CNTs better than the traditional organic solvents. The ionic liquids undergo feeble van der Waals interactions rather than ‘cation-π’ interactions with CNTs. These interactions shielding mechanism also facilitates the dispersion of CNTs in ionic liquids [42]. Recently, Bains et al., revealed the functionalization of MWNTs, i.e., oxidized derivatives of CNTs and graphene using a series of alkylated benzimidazolium based ionic liquids in order to get super-hydrophobic and self-cleaning coatings for robust antibacterial applications in bio-medical

O

O

OH

Furfuralcohol

Ce(IV)/HNO3,PEGMA,AA

DMSO, 24 h

Furfuralcohol

OH O

OH

O O

CNTs-OH-PAA@PEGMA

PEGMA H2C

x O

H2O, N2 CNTs-OH

CNTs

y O O

O O

CH3

AA O

O n

CH3 OH

Figure 3.3  Functionalization of CNTs surface using furfur alcohol, carboxyl group and PEGMA [Adapted with permission from Ref. [40]. Copyright © 2010 Elsevier].

56  Functionalized CNT for Biomedical Applications devices used in hospitals. These ionic liquids are designed such that the N1 position of the benzimidazole contain different chain lengths and the N3 position is left free to additional functionalization with other moieties [43]. Furthermore, poly(ionic liquids) are also used for the functionalization of CNTs in order to impart charged functional group onto CNTs surfaces for enhancing the adsorption capability of the CNTs towards environmental pollutants. This novel functionalization was performed utilizing the combined effect of mussel-inspired chemistry and subsequent surface-initiated reversible addition-fragmentation chain transfer polymerization reactions using 3-n-hexadecyl-1-vinylimidazolium bromide i.e.,[C16VIm+][Br-] ionic liquid as monomer [44]. Functionalization of CNTs with polymer has also gained importance with the aim to impart diverse properties such as response towards environmental stimuli, complex ability with metal ions, enhanced dispersibility in various solvents, high compatibility with polymer matrix, and so on. The polymer chains are chemically bonded to edge or surface of CNTs and give rise to highly stable CNT/polymer composite. For this purpose, the ‘grafting to’, ‘grafting from’ and ‘grafting through’ like chemical methods as well as the traditional free radical polymerization or controlled polymerization methods are mostly exploited [45]. The click chemistry has recently gained importance for the CNTs functionalization too [46]. He et al., discussed the preparation of clicked nanohybrids from polymer coated CNTs and Fe3O4 nanoparticles [47]. The reagents used were (i) H2SO4/HNO3, 90–133 °C, 100 minutes; (ii) SOCl2, 60 °C, one day, (iii) propargyl alcohol, triethyl amine, chloroform, room temperature, one day for obtaining MWNT-C ≡ CH, which was subsequently modified by click grafting to get a polymer containing appreciable number of azide groups (MWNT-pAz), and subsequently, polymer alkyne group was incorporated (MWNT-pAlk). Fe3O4–CRCH and Fe3O4–N3 undergo ‘click’ chemistry reaction on nanotubes coated with polymer and yields MWNTpAz@Fe3O4 and MWNT-pAlk@Fe3O4 as respective magnetic nanohybrids. Similarly, Zhao et al., also utilized click chemistry to synthesize ionic liquid modified MWNTs and then loaded platinum nanoparticle into it. The resulted composite film glassy carbon electron exhibited remarkable analytical performance for detecting tatrazine analytically [48]. New hybrid material of polyhedral oligomeric silsesquioxane and CNTs was prepared by Cho et al., by using click coupling between azide moiety-functionalized polyhedral oligomeric silsesquioxane and alkyne-functionalized MWNTs [49]. Furthermore, this approach was applied for preparing TiO2-based nanohybrids and photo-catalytically active MWNTs [49].

Carbon Nanotubes: Types of Functionalization  57

BC2O BCO2

(150ºC B2O3 solution)

Heat treatment (1000~1800ºC) BC2O

Pyrrolic N

Quaternary N

Pyridinic N

B, N doped CNTF

C

B

N

O

Intensity (Arb. unit)

(Acetone bath)

Densif ication

Nitrogen doping

(d)

(ICP, 50W)

Coagulation

2

4C

5.26

B2O3

2.32

Binding energy (eV)

N2 plasma

Superacid

(at. %)

196 194 192 190 188 186 184 182 BC3

(Chlorosulf ionic bath)

type BC2O

Qu at er na Py r rro y l Py ic rid in ic

Percolation

B1s

B

B BCO2

Boron doping

2O 3

(c)

BC

(b)

Pristine CNTF

Intensity (Arb. unit)

(a)

2O BC O 2 BC

Furthermore, incorporation of heteroatom into the hexagonal carbon lattice of CNTs is termed as heteroatom doping into the walls of CNTs, which is another way of CNTs functionalization. Doping of heteroatoms (like B, N, O, P, S etc.) are generally executed to impart high electronic mobility, good sodium solubility, enhances sodium storage capacity, mass transfer of oxygen, removing water, corrosion inhibition and so on [50, 51]. Hong et al., reported the B and N doping by judiciously using elevated temperature and plasma treatment to obtain macroscopically dense CNTs fibers (CNTFs). It showed synergistic effect of macroscopic densification in combination with heteroatom doping as shown in Figure 3.4 (a) [52]. Hou et al. revealed free standing N-doped CNTs through straightforward pyrolysis of toluene and exhibited good catalytic activity for oxygen reduction reaction with superior selectivity, methanol crossover resistant and stability. Exposed C and N at surface of CNTs provide plenty of active sites [53]. There are various configurations of N doped CNTs, for instances, pyridinic N, pyrrolic N, and graphitic N. The pyridinic N and pyrrolic N are bonded to two carbon atoms

N1s

type

(at. %)

Quaternary 1.22 Pyrrolic 0.16 Pyridinic

0.12

408 406 404 402 400 398 396 394 392

Binding energy (eV)

Figure 3.4  Schematic diagram of (a) boron (B) and nitrogen (N) doping on CNTFs, (b) different types of B (e.g., BCO2, BC2O and BC3) and N (pyrrolic N, pyridinic N and quarternary N) environment created upon doping, X-ray photoelectron spectra of (c) B 1s and (d) N 1s of boron and nitrogen co-doped CNTs. [Reproduced with permission from Ref. [52] Copyright © 2009 Elsevier].

58  Functionalized CNT for Biomedical Applications and situated at the edges, while the quarternary or graphitic N is placed at the core of CNTs substituting the sp2 hybridized carbon atoms as shown in Figure 3.4 (b). Figure 3.4 (c, d) showed B 1s and N 1s X-ray photoelectron spectra of B and N co-doped CNTs. These different configurations having different environments of N which changes the electronic environments of adjacent C atoms give rise to different catalytic activities [54, 55]. Recently, Iizumi et al., reported simple and efficient fabrication methods of O doped CNTs employing Ultra-violet irradiation on the SWNTs thin film with a conventional Ultra-violet ozone cleaner led, which are useful for application in photonic materials owing to oxygen-induced deep trap states [56]. Furthermore, N and P co-doped CNTs are also fabricated through pyrolysis process with combination of rapidly evaporating aerosol droplets [57]. Recently, Hassani and Tavakol revealed the synthesis of S doped CNTs having high S content using chemical vapor deposition technique from sulphur powder, acetylene gas and Fe/CaCO3 as catalysts [58]. Similarly, Kim et al., reported S doping on CNTs (approximately 0.65 % S content) employing chemical vapor deposition technique using dimethyl sulphide as S source. The S doped CNTs exhibited excellent electrochemical performance due to enhanced electrical conductivity and pseudocapacitive property [59]. The co-doping of S and N into the structure of CNTs leads to enhance the electrochemical oxygen reduction activity [60].

3.3.2 Non-Covalent Functionalization of Carbon Nanotubes Non-covalent functionalization deals with interactions occurring between CNTs and molecules. There are numerous approaches reported so far for non-covalent functionalization of CNTs. The host-guest interaction is one of the most significant approaches, wherein, CNTs act as host. The extended π-system of the sidewall of the carbon nanotubes are highly capable to bind with the guest molecules undergoing π-π interactions [61]. Other approaches take advantage of exploitation only through van der Waals interaction between adsorbents and nanotubes. Its advantages depend on the preservation of the properties of the CNT, while its disadvantage is the weak strength between the wrapping/pairing molecules which can reduce the load transfer in the composite [62]. CNTs also have the propensity to intertwine leading to form 3D structure by means of van der Waals interaction. After the CNTs are exfoliated, it facilitates stable dispersion formation in the organic solvents like, phenyl ethyl alcohol or N-methylpyrolidone (NMP) which are capable of efficiently interacting with the CNTs [63, 64]. During sonication, NMP was observed to penetrate the bundles and remain firmly attached with the nanotubes, leading to the enthalpy of the

Carbon Nanotubes: Types of Functionalization  59 mixture being almost zero, just like mixing a negative value of free energy. Similarly, a relatively stable and a good dispersion of CNTs can be obtained using sonication in N, N-dimethylformamide (DMF). This clearly highlights a negligible value of hydrogen bond donation parameter. A high value of hydrogen bond acceptance, i.e., basicity, and very high value for π* (solvatochromic structure) is compulsory. However, to get a good dispersion of CNTs, this condition is not sufficient. The advantage of using non-covalent functionalization is that it doesn’t hamper existing conjugation of π-electrons of the CNT sidewalls; hence, it will not alter the structural property of the material. The interfacial properties of the nanotubes can be tuned by this alternative method, non-covalent functionalization. Various substances like, aryl compounds, polymers, and surfactant can be used for this purpose through π-π stacking interactions and hydrophobic interactions. After this functionalization CNTs are capable of performing previous functions while the solubility factor of CNTs increases dramatically. Aromatic moieties like, porphyrin, pyrene and the derivatives can easily interact with π electrons of CNTs sidewalls through π-π stacking, henceforth; it can be useful for non-covalent functionalization. Dai and co-workers have shown an attractive but general approach for CNTs functionalization using non-covalent pathway using a bifunctional molecules N-succinimidyl -1-pyrenebutanoate followed by subsequent control over immobilizing bio-molecules protein within it. In this process, specificity and high degree of control can be monitored during immobilization The succinimidyl ester get adsorbed onto sidewalls of CNTs surface through a string π-stacking interactions of highly aromatic pyrene moiety with it. The adsorbed succinimidyl ester which is highly reactive in nature, thereby, act as anchor which interacts with the primary or secondary amine groups of the proteins and facilitates the immobilization process as shown in Figure 3.5 (a) [65, 66]. Hecht et al., fabricated a field effect transistor (CNTs/FET)in which the CNTs were functionalized non-covalently with the aid of zinc coated porphyrin like photoactive compound and used for detection of photo induced electron transfer as shown in Figure 3.5 (b). Such type of photosensitive device finds promising applications in photovoltaic devices [66, 67]. In 2008, Hu and co-workers revealed a uniform self-aggregation of a monolayer of highly aromatic pyrene functionalized CdSe nanoparticles over the surface of the CNTs as shown in Figure 3.5 (c). These pyrene CdSe nanoparticles system exhibit effective charge transfer towards CNTs which is the essential criteria of solar cell or heterojunction photovoltaic devices [66, 67]. Furthermore, the non-covalent functionalization of CNTs using [2] catenane tetra cations give rise to bistable monolayer. These [2] catenane

60  Functionalized CNT for Biomedical Applications

(a)

O

(b)

O N

Me Me

Me

O O

N Me

N N

Zn

Me

N

N

N

Me

Me Me

Zn Porphyrin

N-Succinimidyl-1-pyrenebutanoate Pd

Pd SiO2 Si

SWNT

Gate (c)

(d) S

O

O

S

S

CdSe S

O O +N

O

O

S

O

O

O

O

S

S

O

S

O

O

O

S

CdSe

S

S

S

O

O

O

O S

S

S

S

O +ON O

N+

N+

[2]Catenane O

Pyrene/CdSe-SWNT

Figure 3.5  Non covalent functionalization of CNTs, (a) N-succinimidyl-1-pyrenebutanoate-decorated SWNT, (b) zinc porphyrin-coated SWNT/ FET device, (c) SWNT with pyrene/CdSe nanoparticles and a SWNT FET device, (d) catenanes lined up in rows onto SWNTs surface [Adapted with permission from Ref. [66]. Copyright © 2009, American Chemical Society].

Carbon Nanotubes: Types of Functionalization  61 tetra cations are stabilized through self-organization by amphiphilic dimyristoyl phosphatidyl anions as shown schematically in Figure 3.5 (d) [66, 68]. Similarly, there are several organic molecules which have been used to functionalize CNTs viz. 1-pyrenebutanoic acid; succinimidyl ester; 1-(2-anthroquinonylaminomethyl) pyrene; [bis(2-anthaquinonyl)-­ aminomethyl] pyrene; 5,7,12,14-tetramethyldibenzo-1,4,8,11-tetraazacyclotetradeca-3,5,7,10,12,14-hexane; adamentane-pyrene; biotin-pyrene; nitrolotriacetic acid-pyrene; ferrecene; anthacene; anthrarobin; 9,10-dibromoanthracene; 9,10-anthracene-dicarbonitrile; 9-anthracenemethanol; thionine; methyl blue; naphthalene-1-methylphosphonic acid; 1,10-phenanthroline-5,6-dione; triphenylene; porphyrin; hydroxyferriprotoporphyrin and so on [66, 69, 70]. Conjugated polymers are proved to be excellent materials for wrapping in non-covalent functionalization of CNTs. This particularly occurs due to van der Waals as well as π-π stacking interactions occurring between surface of conjugated polymers containing aryl moieties and CNTs. Furthermore, several conjugated poly(m-phenylene)-co-(2,5-ditoxy-p-phenylene) vinylene, poly(2,6-­ pyridinlenevinylene)-co-(2,5-dioctoxy-p-phenylene) vinylene, stilbene like, dendrimers, and poly-(5-alkoxy-m-phenylenevinylene)-co-(2,5dioctoxy-p-­phenylene) vinylene, to explore CNTs functionalization using non-­covalent pathway [70–74]. Surfactants material mainly polymers have also been used for functionalization of CNTs. The surfactant molecule adsorbs physically over CNTs leading to decrease surface tension of the CNTs in turn efficiently prohibit their self-agglomeration. Additionally, electrostatic/steric repulsive force helps CNTs to overcome the van der Waals attractive force. There is a dependency of this method over the strength of the surfactant, chemistry of the medium and polymer matrix. There is a relation between CNTs and surfactants which has been studied well by several scientists for examples (i) non-ionic surfactants, like, polyoxoethylene 8 lauryl [75]; polyoxyethylene octylphenylether (Triton X-100) [76]; (ii) cationic surfactants, like, dodecyl tri-methyl ammonium bromide (DTAB); [77] cetyltrimethyl ammounium 4-vinylbenzoate; [78] and (iii) anionic surfactants, like, sodium dodecyl benzene sulfonate (NaDDBS), sodium dodecyl sulfate (SDS), poly(styrene sulfate) (PSS). The comparative adsorption of surfactants such as NaDDBS, SDS and Triton X-100 leading to stabilization of CNTs has been shown schematically in Figure 3.6. Herein, the parallel adsorption of surfactants onto CNTs influence the stabilization. The alkyl chain of the surfactants is adsorbed in a parallel fashion along the length of the CNTs rather than its diameter. The presence of benzene rings in NaDDBS and Triton X-100 facilitates

62  Functionalized CNT for Biomedical Applications NaDDBS

SO3SO3SO3-

SDS

SO3-Na+

C12H25 SO3-

SO3SO3SO3-

Triton X-100

CH3(CH2)11OSO3-Na+

SO4-

SO4SO4-

SO4-

O(CH2CH2O)N-H

N=approx. 9.5

C8H17

SO4SO4SO4-

Figure 3.6  The schematic representation of how surfactants may adsorb onto the CNTs surface [Reproduced with permission from Ref. [79]. Copyright © 2009, American Chemical Society].

better adsorption which ultimately causes the better dispersion of CNTs. Furthermore, the dispersion capability of NaDDBS is greater than that of Triton X-100. This is attributed to the presence of polar and larger head group (i.e., polyethylene oxide chain) in Triton X-100 which doesn’t stabilize rather lowers the packing density at the CNTs compared to the smaller head group (i.e., sulphur tri oxide anion) of NaDDBS (vide Figure 3.6). Additionally, the electrostatic interaction of sulphur tri oxide anions stabilizes the CNTs through screening Coulomb interactions [79, 80]. However, this surfactant functionalized CNTs are less useful for bio-medical applications. This is due to the toxicity fact of the surfactant, although it can efficiently solubilize the CNTs in medium. And, these surfactants are also known as permeable plasma membrane [81]. For integration of new materials, i.e., biological components in living systems it is necessary to solubilize the CNTs with the component. To achieve the goal several biomacromolecules like, saccharides and polysaccharides, proteins, DNA, engymes, etc. [82–86], were used for functionalizing CNTs in a non-covalent pathway. Furthermore, various bio materials like, n-decyl-β-D maltoside, [87] η-cyclodextrin, [88] γ-cyclodextrin, pullulan, [89] chitosan, and phospholipid-dextran [90] were also exploited for non-covalent functionalization of CNTs. The saccharides and polymers of saccharides do not possess light absorption property in the UV-visible region, therefore, characterization of CNTs in presence of these macromolecules doesn’t hamper its result, especially in photochemical experiments. Moreover, saccharides and polymers of saccharides, i.e., polysaccharides coated CNTs are bio-compatible in nature therefore, may be applied for several bio relevant applications and medicinal purposes. Therefore, the non-covalent functionalization through π-π stacking or van der Waals interaction. All that functionalization is exohedral derivatization i.e., only outer surface will be functionalized. Considering the

Carbon Nanotubes: Types of Functionalization  63 advantage of hollow spherical structural of CNTs, scientists have also shown great interest for endohedral functionalization. In this phenomena, the tubes are filled with atoms or small organic molecules [91–93]. Recently, it has also been seen that there are several discoveries or invention with the aim of non-covalent functionalization of CNTs. Some of the notable ones are the new non-wrapping π-stacking approach for polymer functionalization of CNTs using poly (arylene ethynylene) polymer which comprises of one functionalized moiety to the nanotube in a non-wrapping manner in organic and inorganic solvents. The aromatic or π-conjugated moieties present in the skeleton of the polymer unit undergo parallel π-stacking as the major interaction with the surface of CNTs [94]. Dai et al., disclosed the hydrophilic polymer functionalized SWNTs which are useful for adsorption of amphiphilic molecules to make the SWNTs remain stable in the aqueous suspension and facilitates its drug delivery activities more facile owing to its large surface area available for supramolecular bonding, i.e., π-π stacking between the targeted aromatic drug for delivery and the graphene surface on the nanotubes. Herein, the polymer poly(ethylene glycol) (PEG) and its derivatives which contains –COOH groups was covalently bonded to the sidewall of the nanotubes in order to make the them the active agents for the desired purposes [95]. Strano et al., disclosed non-covalent complexes of SWNTs with biological polymers specifically proteins, polypeptides and polysaccharides which function as sensing polymers. These complexes interact selectively undergoing specific interaction with the analytes like small organic molecules such as sugars, steroids, antigens, as well as polymeric species such as enzymes, proteins, etc. [96]. Tour et al., disclosed the functionalization of exposed sidewall of SWNTs, DWNTs, MWNTs, as well as small diameter carbon nanotubes (SDNTs) in acidic media like fuming sulphuric acid using several organic molecules in presence of NaNO2, AIBN or (t-BuO)2with the aim of obtaining the unbundles well dispersible derivatives of CNTs. Herein, SDNTs are CNTs having diameter of 3 nm [97].

3.3.2.1 Reversibility in Non-Covalent Functionalization Two interesting features are hidden inside non-covalent functionalization. First one is that the functionalization does not hamper the conjugated double bond present in the sidewalls of the CNT. Second one makes this functionalization more interesting and that is the reversibility of the reaction. It is very likely to get the bare CNTs both in aqueous and organic media. Use of different and specific external stimuli is one of the intelligent pathway to prefer for non-covalent functionalization of CNTs [98]. The ability of the

64  Functionalized CNT for Biomedical Applications solvent to desorb from CNTs also depend over the used external stimuli. Many external stimuli can be applied, out of which a few of them are summarized like, solvent, temperature, pH, light and redox property, etc. Many bio molecules can also be used to get reversibility in this functionalization.

3.3.2.2 Solvent Variation in Non-Covalent Functionalization The easier method is the changing of solvent to remove the non-­ covalent functionalization [99]. However, in case of some polymers, the removal of dispersion solvent is bit tough due to strong interaction between the CNTs and the solvents. A simple acetonitrile washing will result to bare CNTs by complete desorption of non-covalent functionalization [100]. When acetonitrile is added in a chlorinated solvent, it not only acts as washing solvent. Moreover, it introduces variation in structures as it possesses solvophobic effect associated with the enhance in polarity with addition of acetonitrile. Another interesting pathway to get proper reversible functionalization is the use of such molecules that can assemble surrounding the nanotubes appearing to be a supramolecular polymer. This fact occurs because hydrogen bonds are effective in keeping nanotubes dispersed with inorganic solvent. However, if the polar solvent is used it disrupts existing bonds and results in precipitation of bare CNTs.

3.3.3.3 pH of the System in Non-Covalent Functionalization It is possible to control the aggregation and disaggregation phase of CNTs by varying the proton concentration. If 1-pyreneacetic acid is added into the solution, the system deprotonates under basic condition due to the presence of pyrene ring within the scaffold. Thereby, it turns out to be facile to go into solution and separates the CNTs along with other carbonaceous impurities in water. After that, in order to get bare CNTs, it must be rinsed thoroughly by distilled water or ethanol. Some polymers can undergo conformational changes due to protonation and deprotonation with the change in pH. Poly(acrylic acid) can be used as dispersing agents for CNTs. Change in pH leads to change in its nature, charge, i.e., ability to from hydrogen bonds. CO2 responsive polymers can also be used to tune the aggregation of CNTs. In presence of CO2, the amidine group of the adsorbed polymer is protonated, and hence the increment of the polymer-polymer electrostatic repulsion results in bare CNTs [101].

Carbon Nanotubes: Types of Functionalization  65

3.3.3.4 Temperature Responsive System in Non-Covalent Functionalization The temperature variation helps in controlling the precipitation of CNTs for instances like folic acid coated CNTs. Reports showed that when it is heated at 80 °C, it results in precipitation of CNTs by peeling of folic acid coating. The only drawback of this method is when it is desired to get back the dispersion, then it is required to apply an external force, for instance, sonication [102]. Wang et al., have established that enthalpically favored conformation of N-(isopropylacrylamide) changes to entropically favored conformation on heating which leads to precipitation of the CNTs. The process seems reversible only by sonicating for 2 minutes at 0 °C, this regenerates the dispersion again [103]. Furthermore, the poly(N-cyclopropylacrylamide) having pendant groups like pyrene stabilizes the aqueous dispersion of CNTs. Interestingly, this dispersion rapidly changes its conformation by reducing the static layer thickness over CNTs leading to precipitation above lower critical temperature [104].

3.4 Conclusion and Future Outlook An appropriate modification or functionalization of the skeleton of CNTs is an important criterion for its effective application in the desired emerging technologies. In this chapter, the most important strategies namely covalent and non-covalent functionalization of CNTs have been outlined and discussed briefly. The typical way of such functionalizations of CNTs helps in improving the dispersibility, stability and processability that ultimately enhances its chemical or electrochemical reactivity as well as various physical properties. The derivatives and composites of CNTs establish a foundation for its exciting and innovative applications. Furthermore, the heteroatom doping are very interesting aspects of CNTs modification. Such functionalization helps in opening new horizon to the CNTs-based composites or hybrids as well as the assembly of other functional advanced materials.

Acknowledgements PB is very thankful to Department of Higher Education, Science & Technology and Biotechnology, Govt. of West Bengal, India for providing financial assistance vide sanction order no. 78(Sanc.)/ST/P/S&T/6G-1/2018

66  Functionalized CNT for Biomedical Applications dated 31.01.2019 and project no. GAP-225612. MM acknowledges Ministry of Tribal Affairs, Govt. of India, New Delhi, India for National Fellowship for Higher Education of Schedule Tribes Students [Erstwhile known as Rajiv Gandhi National Fellowship of Schedule Tribes Students (RGNFST) from University Grants Commission, Govt. of India, New Delhi, India]vide reference no. F1-17.1/2014-15/RGNF-2014-15-ST-JHA-71559/(SA-III/ Website) dated: February 2015. DD acknowledges Department of Science and Technology, Govt. of India, New Delhi, India for DST-INSPIRE fellowship vide reference no. IF160176.

Web Links Web Link1: Bradford research group https://sites.textiles.ncsu.edu/bradford-research-group/research Web Link2: Max-Planck-Institut für Eisenforschung (MPIE) https://www. mpie.de/4413569/carbon-nanotubes Web Link3: North Carolina State University https://www.mse.ncsu.edu/ zhu/research/selected-carbon-nanotubes/ Web Link4: Boies Research Group https://cambridgenanoaerosol.com/ research/

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4 Functionalization Carbon Nanotubes Innovate on Medical Technology Afroz Aslam1*, Jeenat Aslam2, Hilal Ahmad Parray3 and Chaudhery Mustansar Hussain4 Department of Chemistry, Aligarh Muslim University, Aligarh, India Department of Chemistry, College of Science, Taibah University, Yanbu, Al-Madina, Saudi Arabia 3 Department of Pathology, UT Southwestern Medical Center, Texas University Harry Hines Blvd., Dallas, TX, USA 4 Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, NJ, USA 1

2

Abstract

Carbon nanotubes (CNTs) are new nanomaterials with many biomedical applications. CNTs can be utilized  to deliver a variety of therapeutic compounds to disease locations, including biomolecules. Because of their exceptional optical and electrical properties they are also attractive candidates for biomedical applications. CNTs can be functionalized with various functional groups to transfer several moieties for simultaneous targeting, imaging, and therapy. The potential ability to penetrate biological membranes and the minimal toxicity is most promising. This chapter deals with the functionalized carbon nanotubes (f-CNTs) in all medical fields. Keywords:  Functionalized carbon nanotubes, biomaterials, medical applications, tissue engineering, infectious diseases treatment, cancer treatment

4.1 Introduction The most significant advantage of nanotechnology is the ability to produce advanced structures with improved ability to translocate through *Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (75–94) © 2023 Scrivener Publishing LLC

75

76  Functionalized CNT for Biomedical Applications cell membranes and higher solubilization, stability, and bioavailability of biomolecules, hence improving their delivery efficiency. Nanotechnology holds potential for various biomedical applications, including bioimaging and targeted delivery of biomacromolecules into cells. Aono and Ariga [1–3] have introduced nanoarchitectonics, an emerging new idea in nanotechnology. This innovative concept has ushered in a new era in material creation, an innovative technology that combines numerous elements and effects to create functional nanomaterials and nanosystems. Materials, mechanics, electronics, packaging, cosmetics, optical devices, textiles, and solar cells are just a few domains where this technology might be used. This technology has recently been broadened to include biomedical applications such as cultivation, detection, diagnostics, imaging, and various cancer therapies [4, 5]. The primary goal of developing nano carrier-based drug delivery structures is to increase the therapeutic efficacy of therapeutically active chemicals while lowering their toxicity. Liposomes, which are spherically shaped vesicle nanocarriers, are commonly used. CNTs, on the other hand, are cylindrical carbon atom molecules. CNTs are graphene sheets rolled into a seamless cylinder with a high aspect ratio with diameters as small as 1 nm and a length of several micrometers, which can be capped or open-ended. Single-walled carbon nanotubes (SWNTs) are generated from a single graphene sheet, whereas multi-walled carbon nanotubes (MWNTs) are made from many graphene sheets [6, 7] (Figure 4.1). Because of their typical chemical and physical properties and possible applications in a wide range of fields, from electronic and sensors devices to nanocomposite materials of high strength and low weight, these allotropes of carbon have generated intense interest since their discovery in 1991 by Iijima [6]. Pristine CNTs are not soluble. CNTs bio applications were only possible following the invention of techniques to functionalize these molecules with organic groups and make them soluble. Due to their large surface area, they can adsorb or conjugate with a broad range of medicinal compounds. CNTs can thus be functionalized to improve their aqueous phase dispersibility or offer the proper functional groups to combine with the required target tissue or therapeutic material to elicit a therapeutic effect. CNTs may aid in the therapeutic molecule penetration into the target cell to treat diseases [8–11]. A current instance of CNTs with a range of functional groups is essential to cancer therapy [12] (Figure 4.2).

CNTs Innovate on Medical Technology  77 (a)

(b)

(c)

Figure 4.1  Structure of (a) a single-walled carbon nanotube (SWCNT), (b) a doublewalled carbon nanotube (DWCNT), and (c) a multi-walled carbon nanotube (MWCNT) [7] (open access source).

Cancer therapy hyperthermia

Electromagnetic Wave absorption Diagnosis of cancer

Multi-walled Carbon Nanotube

High Conductivity

Excellent mechanical strength

Biomaterial by CNT composite

DDS applications

Large specific volume at nano size

Good compatibility, Adhesion

Scaffold for regeneration

Figure 4.2  Characteristics of Carbon nanotubes and medical applications [13]. (open access source).

78  Functionalized CNT for Biomedical Applications

4.2 Functionalization CNTs for Biomedical Applications CNTs are generally dispersible and insoluble substances; thus, it’s critical to enhance their surface qualities for better solubilization, dispersion, cytotoxicity reduction, and biocompatibility. CNT surfaces could be modified to increase their biocompatibility and reduce their cytotoxicity in biological systems for biomedical applications by improving their solubility in water, serum, and other solvents [14]. The surface chemistry of CNTs and the method of purification and functionalization have a substantial impact on their biological activities and cytotoxic effects. Based on the behavior of the biomolecule connected to CNT, the functionalization process may be categorized into two types: covalent (chemical bond formation) and noncovalent (physisorption) attachments [15, 16]. Carbon nanotubes’ biocompatibility and solubility are the most significant characteristics of their utilization in biomedical applications. Purification of carbon nanotubes through covalent functionalization using a multistep acid treatment should increase solubility and lower toxicity [17]. Different purification procedures can make CNTs more soluble, exposing particular charged groups and lowering cytotoxicity [18]. Acid-oxidized, short, carboxylated CNTs with high aqueous dispersions and hydrophilic surfaces were less lethal and biocompatible in mice [19]. Human monocyte-derived macrophage cells were used to study the uptake of acid-treated, water-soluble single walled CNTs (SWCNTs). Purified CNTs (P-CNTs) were found inside the cytoplasm and lysosomes with no effect on the structure or cell viability compared to unpurified CNTs (UP-CNTs). Functionalized SWCNTs have also been utilized on human cells, implying that eliminating hazardous toxins from carboxylated SWCNTs is essential for their progress in pharmaceutical applications [20]. The addition of chemical groups, for example –CO,-OH, and -COOH, to CNTs boosts their O2 concentration, reducing P-CNT cytotoxicity [21, 22]. Nonviral vectors made of oxidized  ultrashort SWCNTs have been employed to transfer oligonucleotide molecules to human macrophages without causing cytotoxicity [23]. It was recently discovered that scattered SWCNTs are pretty safe in cytotoxicity and that isolated and pure SWCNTs are incapable of causing acute cell death [24]. Covalent functionalization of CNTs is a better approach which depends on the degree of functionalization [21] and improves CNT biocompatibility while reducing cytotoxic effects. Noncovalent functionalization is another process to modify the CNT’s surface. Surface adsorption onto the sidewalls of CNTs, hydrogen

CNTs Innovate on Medical Technology  79 Functionalization of CNTs

Non – Covalent Methods

Convalent Methods

Side wall

Ends and Defects

Polymer wrapping

Surface attachments of surfactants

Figure 4.3  Covalent and Non-covalent functionalization methods of CNTs. “Reprinted with permission from [26]. Copyright (2018) Elsevier.”

bonding, electrostatic interactions, stacking, and the van der Waals forces are all examples of noncovalent interactions between CNTs and other molecules. CNTs become more water-miscible due to noncovalent techniques, making them less poisonous [25]. To achieve biocompatibility, various biomolecules, polymers, and surfactants have been employed to functionalize CNTs noncovalently. The interaction between graphitic and pyrene surface of CNTs has also been used to coat porphyrin derivatives, and fluorescein isothiocyanate (FITC) terminated poly(ethylene glycol) (PEG) chains onto the surface of CNT, resulting in increased biocompatibility and reduced toxicity [25, 26] (Figure 4.3).

4.3 Potential Applications of CNTs in Cancer Therapy Due to their unique features, CNTs have been comprehensively studied in delivering targeted drugs for cancer therapy. Owing to their intrinsic optical properties, CNTs can not only be used as drug carriers to deliver numerous anticancer medicines, but they can also be used as excellent phototherapy mediators. CNTs’ multifunctionality allows them to be used in various cancer treatments. Many anticancer therapy techniques currently aim to target tumor cells and the environment in which they thrive. Tumor cells can be targeted directly to destroy their parenchyma. In contrast, the tumor microenvironment (TME) can be targeted to prevent tumor development and metastasis by disrupting their living conditions, indirectly killing tumor cells.

80  Functionalized CNT for Biomedical Applications

4.3.1 Anti-Tumor Immunotherapy Many studies have indicated that CNTs utilized as carriers are effective in anticancer immunotherapy [27]. This treatment stimulates the patient’s immune system to attack the malignant tumor cells. This stimulation can be induced by delivering a therapeutic antibody or a cancer vaccine as a medicine. Even though  several medical trials of immunotherapy have shown potential outcomes in cancer treatment [28, 29], immune therapy’s low therapeutic efficiency remains a significant concern [30]. CNTs have been approved as vaccine delivery systems due to properties such as lower immunogenicity than standard protein carriers, the capability to permeate the cell membrane without no causing damage, and a more excellent immune response when paired with an antigen [31, 32]. In a mouse model with the H22 liver tumor, a combination of multiwalled CNTs (MWCNTs) and tumor lysate protein (tumor cell vaccination) can significantly and specifically improve the efficacy of anticancer immunotherapy. Because of the high avidity of antigen on the surface and the negative charge, CNTs linked to tumor immunogens can function similarly to professional antigen-presenting cells (such as mature dendritic cells) in delivering cancer antigens to immunological effect or T cells in vitro [33]. CNTs are also said to activate the complement system and have adjuvant effects. The mechanism, however, is uncertain [34, 35].

4.3.2 Anti-Tumor Hyperthermia Therapy CNT-based hyperthermia therapy has recently been recommended as a promising cancer potential treatment. SWCNTs have significant absorption in the near-infrared spectrum (NIR; 700–1100 nm) [36]. Biological systems, by coincidence, are translucent to a similar spectral window. Due to the small autofluorescence setting of tissues and cells can be employed for optical imaging of nanotubes inside living cells [37]. Its simplicity and stability make it preferable to radioactive labels or traditional fluorophores for detecting the pharmacorekinetics of SWCNTs in vivo [38]. Near-Infrared Light (NIR) light mixed with a laser pulse could rupture the endosome and facilitate the release of DNA carried by SWCNTs in under-regulated settings [39]. Local thermal ablation of tumor cells triggered via extreme heating of SWCNTs trapped in tumor cells is a comparable application of NIR absorbance. The photothermal effect can induce the local thermal ablation of tumor cells by excessive heating of SWCNTs shackled in tumor cells such as pancreatic cancer. Recently, some progress has been made in the technique, demonstrating clinical possibility [39–41].

CNTs Innovate on Medical Technology  81

4.3.3 Anti-Tumor Chemotherapy Chemotherapy, along with surgery and radiation, is now an essential part of treating advanced cancers. Two significant difficulties have vulnerable traditional chemotherapy: low chemotherapeutic agent specificity, which results in severe systemic toxicity, and drug resistance (acquired or intrinsic to malignant cells), which results in small efficiency. CNTs have been investigated as new drug delivery vehicles with low toxicity and immunogenicity. CNTs have been used to transport a variety of medicinal compounds, with some outcomes performing those achieved with traditional carriers [42–44]. When utilized in anticancer chemotherapy, CNTs’ high surface area permits the effective loading of chemotherapeutic drugs [45]. Drug-loaded CNTs (also known as macromolecular agents) can extravagate into tumor  tissues over time, resulting in a concentration in the tumor that is many times higher than in the plasma due to the better permeability and retention impact in solid tumors [46]. Dai’s team discovered that 13wt% of the PEGylated SWCNTs injected into cancer accumulated [47]. The same group combined paclitaxel with SWCNTs in a mouse 4T1 breast cancer model. It administered the SWCNT–paclitaxel combination in-vivo to obtain superior efficiency in suppressing tumor development while avoiding apparent adverse effects on normal organs [48]. SWCNTs’ improved therapeutic efficiency and fewer side effects could be explained by their more extended blood circulation, slower drug release, and stronger tumors absorption (Tenfold larger than Taxol). Another broadly

Blood vessel

Folate receptor Folic acid PtIV prodrug

H O N O H3N CI O Pt O H3N CI O N O H

Nucleus

O

O

CI O H3N Pt CI O H3N O HO O

H N

O

O O

H HO N O

O N H

O

O N H

N

O

O

Folate receptor (FR) H HO OO N N N OH O N H N H N N NH2

OH N

N N NH2

Figure 4.4  One example of CNT used as a drug carrier. Cisplatin is covalently ligated to superficially oxidized CNTs as an effective anti-tumor agent, and a folic acid molecule is further coupled to the cisplatin as a targeting molecule. The large surface area of CNTs makes it possible to carry more cisplatins into tumor cells. “Reprinted with permission from [49]. Copyright (2008) American Chemical Society.”

82  Functionalized CNT for Biomedical Applications applied anticancer drug, cisplatin, has been employed as a ‘longboat delivery system’ with SWCNT, resulting in increased uptake in cancerous cells [49]. Antibodies to antigens overexpressed on the cancerous cell surface or ligands that target particular receptors on the tumor cell surface can direct CNTs to the diseased surface of the cell (Figure 4.4). CNTs can be taken up by cells before chemotherapeutic medications are cleaved off, allowing for targeted administration. The previously used targeted molecule was Folic acid [49–52].

4.3.4 Other Cancer Treatment Strategies Immunotherapy is a treatment option for incurable cancers. Because of its immense potential in targeting tumors, anticancer immunotherapy using CNTs has been intensively investigated in connection with the novel application of immunotherapy to cancer therapy. McDevitt and colleagues [53] used tumor-specific monoclonal antibodies attached to SWCNTs, a metal-­ ion chelate to carry or deliver a radioactive metal ion, and fluorescent chromophores to visualize their location to target lymphoma. SWCNTs were also employed by Ruggiero et al. [54] to bind tumor-­associated angiogenesis-targeting antibodies E4G10 and radioactive metal ion chelates. Due to their dual activities in targeting and radio immune therapy, this formulation could simultaneously reduce tumor volume and raise the survival rate of LS174T tumor-bearing model mice. Using a subcutaneous injection method, Meng et al. [55] investigated the immunological responses generated by oxidized MWCNTs in hepatocarcinoma-bearing mice. The injection of CNTs resulted in a significant increase in the secretion of inflammatory cytokines and the stimulation of macrophage phagocytosis. These effects stimulate the synthesis of the host immune system and reduce tumor growth. As a result, these results indicate that CNTs can cause anticancer activity in the host by eliciting a tumor-specific immune response.

4.4 Treatment of Central Nervous System Disorders Delivering medications to the central nervous system remains a crucial challenge in the anticancer drug delivery system for treating tumors of the central nervous system due to the blood-brain barrier. Acetylcholine is a cholinergic nervous system neurotransmitter linked to high-level neurological processes like thinking, memory, and learning. Acetylcholine levels

CNTs Innovate on Medical Technology  83 in Alzheimer’s brain neurons diminish due to the synthesis difficulty, causing memory, thinking, and learning impairments. Providing acetylcholine to neurons could help patients with Alzheimer’s disease demonstrate their cognitive abilities. Because acetylcholine is a molecule with high polarities, it is hard to pass by the blood-brain barrier; there has been no technique to transport it into the brain. The Raman spectrum indicated the adsorption of acetylcholine on SWCNT in a recently developed drug delivery system, albeit it remained unclear whether acetylcholine was adsorbed in the tubes or on the surfaces SWCNT. This approach effectively supplied acetylcholine into brain neurons via axoplasm neurite transformation (Figure 4.5) and notably enhanced Alzheimer’s disease model animals [56, 57]. This delivery system was the first to use nanocarriers to transport medications into central nervous system neurons, opening the mode for drug delivery systems to be used to treat malignancies of the central nervous system. In the in vivo anticancer drug delivery system, the results of CNT-based drug delivery were compared to numerous commercially available formulations. The study indicates that carbon nanotubes had significant benefits over other nanomaterial-based drug delivery technologies. The efficiency and toxicity of doxorubicin-loaded CNTs with doxorubicin-loaded nanoliposomes were compared by Liu et  al. [58]. Though, a significant conclusion on whether CNTs are the most excellent

CNTs for various neurological disease therapeutics as inherent medication Carbon nanotubes

In vivo

Neural stimulation

In vitro

Neuronal differentiation

Neuro de/regeneration

Functional neurosurgery Stroke

Neural interface

In vitro

Neuro oncology

Drug abuse addiction

CNTs

In vivo

Figure 4.5  CNTs for various neurological diseases therapeutics as inherent medication [57] (open access source).

84  Functionalized CNT for Biomedical Applications carriers in anticancer drug delivery systems has yet to be reached because several other anticancer drug delivery systems based on other nanomaterials, for example, cationic polymers [59], fibrinogens [60], and PVP/PVAL nanoparticles [61], have also demonstrated immense promise as drug carriers for cancer treatments. Further contrast studies are needed to confirm CNTs’ role as drug carriers in anticancer therapy. Moreover, the fact that the diameter, volume, and length of CNTs are not adequately managed in research, nor is there any accurate information on their dimension sharing, would not present a significant threat to the reproducibility of pharmacokinetic results. These issues will hinder the commercialization of CNT-based drug delivery technologies. To the best of our understanding, no such system has gone through a clinical study. Numerous challenges must yet be defeated before CNT-based drug delivery systems can be used in actual medicine.

4.5 Treatment of Infectious Diseases Antibiotics could be used for f-CNTs differently. The antimycotic drug amphotericin B (AmB) is used to treat fungal strains that are very resistant [62]. It is, however, of restricted utility because, due to its low water solubility and penchant for aggregating and generating holes in the cell membrane, it is exceedingly toxic to mammalian cells. We reasoned that conjugating AmB to CNTs might alter its properties in terms of antimycotic efficiency and toxicity [63]. Cytotoxicity of f-CNT1against mammalian cells was the first issue we addressed. While AmB is indeed very poisonous at 10 g/mL, causing 40% cell death, CNT-conjugated AmB5 was not hazardous when utilized at maximum concentrations (up to 40 g/mL, equivalent to AmB concentration attached to 10 g/L tubes). The capability off-CNT1 containing both fluorescein and AmB to penetrate cells was then studied (Figure 4.6). The detection off-CNT 1 in cells is enabled by this component. This component allows for the identification of f-CNT 1 in enclosures. The fluorescence was evident inside the cell compartments. During the same time frame, toxicity against fungi and yeasts increased. According to preliminary results, f-CNT 2 is also active on AmB-resistant strains. The cause for this improved therapeutic efficacy is unknown. A more in-depth investigation is undertaken to determine whether the action mechanism off-CNT 2 is comparable to AmB or varies in specific ways, thereby explaining the decreased toxicity to mammalian cells.

CNTs Innovate on Medical Technology  85 OH

OH

HO H N

O

O OH

OH

OH

OH

O

OH

O

O

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O

OH

O

NH2

N OH

O

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F-CNT-1

COOH

O

S NH

HO

NH O

N H

O

NH O

OH

OH

HO

O

O OH OH

OH OH O

OH

O O

O O

OH NH2

N OH

F-CNT-2

Figure 4.6  Molecular structures of the carbon nanotube conjugated with different therapeutic agents.

4.6 CNTs-Based Transdermal Drug Delivery CNTs can successfully transfer active drug molecules through the skin because they are composed of an adsorptive material with a large surface area [64]. For hydrophobic drugs, CNTs can provide a high loading and improved transdermal penetration [65]. Because of the electro-conductive qualities of CNTs and the potential for regulated release, iontophoresis is also a viable option. The use of a CNT-based membrane can improve the application of iontophoresis. To improve the electrical sensitivity of the transdermal delivery method, MWCNTs were mixed with a matrix made of polyethylene oxide and pentaerythritol triacrylate polymers [66].

86  Functionalized CNT for Biomedical Applications The amount of drug released rises as the electric voltage increases implies that drug release is controlled by the dissolution of polyethylene oxide in the polymer network, which CNTs support.

4.7 f-CNTs for Vaccination Another type of CNTs-based therapy candidate uses synthetic peptides to stimulate the immune system. Adding B and T cell peptide epitopes to functionalized nanotubes can result in a multivalent system capable of generating a significant immunological response [67, 68]. Chemoselective connections can be made between peptides and tubes [69]. This methodology depends on the production off-CNTs with a maleimide group that quickly combines with peptides that include a cysteine residue on one end. The thiol group of cysteine attaches to the maleimide selectively, forming a stable covalent bond. This method can fully deprotect and characterize the peptide generated through the solid-phase synthesis before ligation to the nanotubes. By connecting a B cell epitope from a foot-and-mouth disease virus coat protein to a B cell epitope, f-CNT 3 and 4 (Figure 4.7) was created (FMDV). The amount of peptide around the nanotubes in the two conjugates differs, doubled for f-CNT 4 utilizing a lysine branch. The antigenic and immunogenic properties of these conjugates were examined. Compared to the nonconjugated peptide, f-CNTs 3 and 4 induced significant antibody responses. Furthermore, the produced antibodies were able to neutralize the virus, demonstrating the potential of CNTs as a candidate for a synthetic vaccine—Figure 4.8 [70].

4.8 Application of f-CNTs in Tissue Engineering CNTs have mainly been employed as reinforcement filler in polymer-based scaffolds in tissue engineering, increasing electrical and mechanical properties and permitting the production of stands for cardiac, neural, and bone tissue engineering. The addition of CNTs in the polymer matrix can improve the conductivity of nerve-related scaffolds and improve the responses of nerve cells. It also enhances the cardiac-related scaffolds’ elastic strength, conductivity, and biological reactions. CNTs are employed to strengthen both the mechanical and biological aspects of bone tissue engineering. Non-toxic, greener, and safer CNTs and polymeric composites are still predicted in the future. Research into how CNTs generate toxicity is still needed to utilize better the potential factors that cause gen

CNTs Innovate on Medical Technology  87 O

S

O

N

F-CNT-3 O

O

VDMF-Cys-Ac O

Ac-Cys-FMDV

N

NH

O

O

N

O

HN

O

N

S

O O NH

O O

O

S

HN

N

VDMF-Cys-Ac

O

O

O

O

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S VDMF-Cys-Ac

O

S Ac-CysO FMDV

S

N O

O

Ac-Cys-FMDV

N

F-CNT-4

O

O

N H HN

N

N

O H N

O

NH O

O

Figure 4.7  Molecular structures of the carbon nanotube conjugated with different therapeutic agents.

cytotoxicity. Many research investigations focus on the early stages of cell development in vitro; therefore, long-term in vivo studies containing the degradation of the polymeric material that leads to the CNTs release must be examined. The present research examines the small proportion of CNTs in scaffolds because of production constraints and toxicity while balancing the scaffold’s electrical, mechanical, and biological capabilities. As a result, more significant CNTs concentrations in the polymer matrix are predictable. Additive manufacturing (e.g., vat-photopolymerization and extrusion-based methods) has been utilized  to construct scaffolds with controlled architecture that outperformed 2D membranes or electrospun

88  Functionalized CNT for Biomedical Applications CNTs in vaccination

Specific antibodies

B cells Antigen Antibody

FCR

• First signal • Second signal INF’Y and IL-17

T cells

CD4+ and T cell activation

ls

esse

dv Bloo

Figure 4.8  Schematic representation of CNTs mediated peptide delivery for immunization, which showed that vaccination by peptide leads to activated T cell response, which is responsible for specific antibodies. “Reprinted with permission from [70]. Copyright (2019) Elsevier.”

meshes in mimicking the 3D environment of biological tissues. Different advanced manufacturing technologies will be utilized in the future to construct hierarchical and multi-material scaffolds that closely mimic the natural atmosphere for differentiation and cell proliferation to convert CNTs involving polymer composite scaffolds into clinical achievement.

4.9 Conclusion CNTs are incredibly fascinating to medical candidates because they have chemically modifiable surfaces with variable lengths and large surface area, besides distinctive physical properties. Because of their potential as drug

CNTs Innovate on Medical Technology  89 carriers in targeted delivery, CNTs promise to detect and treat resistant diseases, for example, central nervous system (CNS) disorders, cancer, and infectious diseases. They’re fascinating to study targets in tissue engineering because of their unique mechanical properties. CNTs are currently being examined for potential toxicity; however, they are generally thought to be a safer option than other nanomaterials like quantum dots or other medicine carriers like virus carriers. As a result, CNTs still need to be tested in vivo for their ADME profile before being employed safely and efficiently in nanomedicine.

Important Websites https://pubs.acs.org/doi/10.1021/acs.jmedchem.5b01770 https://books.google.com.sa/books/about/Carbon_Nanotubes_for_ Biomedical_Applicat.html?id=irEuu80Rg3IC&printsec=frontcover&source=kp_read_button&hl=en&redir_esc=y#v=onepage&q&f=false https://www.sciencedirect.com/science/article/pii/S0928493118320101 https://en.wikipedia.org/wiki/Potential_applications_of_carbon_nanotubes

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92  Functionalized CNT for Biomedical Applications 35. Bottini, M., Bruckner, S., Nika, K., Bottini, N., Bellucci, S., Magrini, A., Bergamaschi, A., Mustelin, T., Multi-walled carbon nanotubes induce T lymphocyte apoptosis. Toxicol. Lett., 160, 2, 121–126, 2006. 36. O’connell, M.J., Bachilo, S.M., Huffman, C.B., Moore, V.C., Strano, M.S., Haroz, E.H., Rialon, K.L., Boul, P.J., Noon, W.H., Kittrell, C., Ma, J., Band gap fluorescence from individual single-walled carbon nanotubes. Science, 297, 5581, 593–596, 2002. 37. Welsher, K., Liu, Z., Daranciang, D., Dai, H., Selective probing and imaging of cells with single-walled carbon nanotubes as near-infrared fluorescent molecules. Nano Lett., 8, 586–590, 2008. 38. Cherukuri, P., Gannon, C.J., Leeuw, T.K., Schmidt, H.K., Smalley, R.E., Curley, S.A., Weisman, R.B., Mammalian pharmacokinetics of carbon nanotubes using intrinsic near-infrared fluorescence. Proc. Natl. Acad. Sci., 103, 18882–18886, 2006. 39. Kam, N.W.S., O’Connell, M., Wisdom, J.A., Dai, H., Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci., 102, 11600–11605, 2005. 40. Hirsch, L.R., Stafford, R.J., Bankson, J.A., Sershen, S.R., Rivera, B., Price, R.E., Hazle, J.D., Halas, N.J., West, J.L., Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci., 100, 13549–13554, 2003. 41. Chakravarty, P., Marches, R., Zimmerman, N.S., Swafford, A.D.E., Bajaj, P., Musselman, I.H., Pantano, P., Draper, R.K., Vitetta, E.S., Thermal ablation of tumor cells with antibody functionalized single-walled carbon nanotubes. Proc. Natl. Acad. Sci., 105, 8697–8702, 2008. 42. Klumpp, C., Kostarelos, K., Prato, M., Bianco, A., Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics. Biochim. Biophys. Acta Biomembr., 1758, 404–412, 2006. 43. Bianco, A., Kostarelos, K., Prato, M., Applications of carbon nanotubes in drug delivery. Curr. Opin. Chem. Biol., 9, 674–679, 2005. 44. Pastorin, G., Crucial functionalizations of carbon nanotubes for improved drug delivery: A valuable option? Pharm. Res., 26, 746–749, 2009. 45. Liu, Z., Sun, X., Nakayama-Ratchford, N., Dai, H., Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery. ACS Nano, 1, 50–56, 2007. 46. Maeda, H., Bharate, G.Y., Daruwalla, J., Polymeric drugs for efficient tumor-targeted drug delivery based on EPR-effect. Eur. J. Pharm. Biopharm., 71, 409–419, 2009. 47. Liu, Z., Cai, W., He, L., Nakayama, N., Chen, K., Sun, X., Chen, X., Dai, H., In vivo biodistribution and highly efficient tumor targeting of carbon nanotubes in mice. Nat. Nanotechnol., 2, 47–52, 2007. 48. Liu, Z., Chen, K., Davis, C., Sherlock, S., Cao, Q., Chen, X., Dai, H., Drug delivery with carbon nanotubes for in vivo cancer treatment. Cancer Res., 68, 6652–6660, 2008.

CNTs Innovate on Medical Technology  93 49. Dhar, S., Liu, Z., Thomale, J., Dai, H., Lippard, S.J., Targeted single-wall carbon nanotube-mediated Pt (IV) prodrug delivery using folate as a homing device. J. Am. Chem. Soc., 130, 11467–11476, 2008. 50. Kam, N.W.S., O’Connell, M., Wisdom, J.A., Dai, H., Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci., 102, 11600–11605, 2005. 51. Bhirde, A.A., Patel, V., Gavard, J., Zhang, G., Sousa, A.A., Masedunskas, A., Leapman, R.D., Weigert, R., Gutkind, J.S., Rusling, J.F., Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery. ACS Nano, 3, 2, 307–316, 2009. 52. Welsher, K., Liu, Z., Daranciang, D., Dai, H., Selective probing and imaging of cells with single-walled carbon nanotubes as near-infrared fluorescent molecules. Nano Lett., 8, 586–590, 2008. 53. McDevitt, M.R., Chattopadhyay, D., Kappel, B.J., Jaggi, J.S., Schiffman, S.R., Antczak, C., Njardarson, J.T., Brentjens, R., Scheinberg, D.A., Tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes. J. Nucl. Med., 48, 1180–1189, 2007. 54. Ruggiero, A., Villa, C.H., Holland, J.P., Sprinkle, S.R., May, C., Lewis, J.S., Scheinberg, D.A., McDevitt, M.R., Imaging and treating tumor vasculature with targeted radiolabeled carbon nanotubes. Int. J. Nanomed., 5, 783–802, 2010. 55. Meng, J., Yang, M., Jia, F.M., Kong, H., Zhang, W.Q., Wang, C.Y., Xing, J.M., Xie, S.S., Xu, H.Y., Subcutaneous injection of water-soluble multi-walled carbon nanotubes in tumor-bearing mice boosts the host immune activity. Nanotechnology, 21, 145104–145112, 2010. 56. Xiang, C., Zhang, Y., Guo, W., Liang, X.J., Biomimetic carbon nanotubes for neurological disease therapeutics as inherent medication. Acta Pharm. Sin. B, 10, 2, 239–248, 2020. 57. Yang, Z., Zhang, Y., Yang, Y., Sun, L., Han, D., Li, H., Wang, C., Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer’s disease. Nanomedicine, 6, 427–441, 2010. 58. Liu, Z., Fan, A.C., Rakhra., K., Sherlock, S., Goodwin, A., Chen, X., Yang, Q., Felsher, D.W., Dai, H., Supramolecular stacking of doxorubicin on carbon nanotubes for in vivo cancer therapy. Angew. Chem. Int. Ed. Engl., 48, 41, 7668–7672, 2009. 59. Nimesh, S., Gupta, N., Chandra, R., Cationic polymer-based nanocarriers for delivery of therapeutic nucleic acids. J. Biomed. Nanotechnol., 7, 504–520, 2011. 60. Rejinold, N.S., Muthunarayanan, M., Chennazhi, K.P., Nair, S.V., Jayakumar, R., Curcumin loaded fibrinogen nanoparticles for cancer drug delivery. J. Biomed. Nanotechnol., 7, 521–534, 2011. 61. Terence, M.C., Faldini, S.B., Miranda, L.F., Miranda, F., Munhoz Jr., A.H., Castro, P.J., Preparation and characterization of a polymeric blend of PVP/

94  Functionalized CNT for Biomedical Applications PVAL for use in drug delivery system. J. Biomed. Nanotechnol., 7, 446–449, 2011. 62. Zotchev, S.B., Polyene macrolide antibiotics and their applications in human therapy. Curr. Med. Chem., 10, 211–223, 2003. 63. Wu, W., Wieckowski, S., Pastorin, G., Benincasa, M., Klumpp, C., Briand, J., Gennaro, R., Prato, M., Bianco, A., Targeted delivery of Amphotericin B to cells using functionalized carbon nanotubes. Angew. Chem. Int. Ed., 44, 6358–6362, 2005. 64. Ilbasmis-Tamer, S. and Degim, I.T., A feasible way to use carbon nanotubes to deliver drug molecules: Transdermal application. Expert Opin. Drug Deliv., 9, 8, 991–999, 2012. 65. Degim, I.T., Burgess, D.J., Papadimitrakopoulos, F., Carbon nanotubes for transdermal drug delivery. J. Microencapsul., 27, 669–81, 2010. 66. Im, J.S., Bai, B.C., Lee, Y.S., The effect of carbon nanotubes on drug delivery in an electro-sensitive transdermal drug delivery system. Biomaterials, 31, 1414–19, 2010. 67. Pantarotto, D., Hoebeke, J., Graff, R., Partidos, C.D., Briand, J.-P., Prato, M., Bianco, A., Synthesis, structural characterization and immunological properties of carbon nanotubes functionalized with peptides. J. Am. Chem. Soc., 125, 6160–6164, 2003. 68. Pantarotto, D., Partidos, C.D., Hoebeke, J., Brown, F., Kramer, E., Briand, J.-P., Muller, S., Prato, M., Bianco, A., Immunization with peptide-functionalized carbon nanotubes enhance virus-specific neutralizing antibody responses. Chem. Biol., 10, 961–966, 2003. 69. (a) Goodman, M., Felix, A., Moroder, L., Toniolo, C., Methods of Organic Chemistry, vol. E22b, Houben-Weyl, Thieme, Stuttgart, 2002. (b) Muller, S., Laboratory Techniques in Biochemistry and Molecular Biology, vol. 28, S. Pillai, and P.C. van der Vliet, (Eds.), pp. 79–131, Elsevier, Amsterdam, 1999. 70. Maheshwari, N., Muktika, T., Namrata, S., Piyush, G., Mukesh, C.S., Pran, K.D., Rakesh, K.T., Functionalized carbon nanotubes for protein, peptide, and gene delivery, in: Biomaterials and Bionanotechnology, Advances in Pharmaceutical Product Development and Research, pp. 613–637, Academic Press, 2019.

Part 2 FUNCTIONALIZED CARBON NANOTUBES: CURRENT AND EMERGING BIOMEDICAL APPLICATIONS

5 Functionalized Carbon Nanotubes: Applications in Biosensing N. Palaniappan1*, Nidhi Vashistha1 and Ruby Aslam2 School of Chemical Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India 2 Corrosion Research Laboratory, Department of Applied Chemistry, Aligarh Muslim University, Aligarh, India 1

Abstract

The functionalized carbon nanotubes are used in several applications because of their excellent physicochemical properties. The current transition metal oxide decorated CNT materials have shown significant sensing. We have discussed different organic hetero atoms and nanoparticles incorporated into CNT and their sensing behaviors. However, zinc oxide functionalized CNT showed excellent glucose-­ sensing due to the nonbonding electron involved in the accept and donate mechanism. Hence, the porous nanoparticles decorated carbon nanotubes for sensing ammonia at room temperature. However, functionalized carbon nanotubes could be revolutionizing the biomedical sensing application at a low cost in the future. Keywords:  Sensor, functionalized carbon nanotubes, biomedical

5.1 Introduction Since the finding of carbon nanotubes (CNTs) in 1991 by Iijima, it has gathered intense attention from the scientific community globally [1, 2]. For the past two decades, enormous advances have been made in both the understanding of the properties of CNTs and in exploring the methods for synthesis. CNTs exhibit outstanding thermal, electrical mechanical, and optical properties. CNTs can find a wide range of applications in different areas including composite materials (fillers or coatings), additives *Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (97–116) © 2023 Scrivener Publishing LLC

97

98  Functionalized CNT for Biomedical Applications to polymers and catalysts, flat displays, cathode rays of lighting components, lithium battery anodes, absorption and screening of electromagnetic waves, conversion of energy, hydrogen storage, telecommunication devices, nanoprobes, ultrasensitive chemical and biosensors, supercapacitors, and transparent conductive membranes, etc., [3, 4]. CNT structure can be represented as an elongated hollow cylinder constituting one or many hexagonal graphite sheets folded into a tube. Each carbon atom in hexagonal arrangement shares an sp2 hybridization (C-C bond) with its three nearby carbon atoms [5]. Although, the circular curvature of the tube can lead to quantum confinement and rehybridization of the σ-π orbital, where three σ bonds are marginally out of the plane; to compensate for this, delocalization of the π orbital is more to the outer side of the tube. This causes nanotubes thermally and electrically to be more conductive, mechanically more robust, and enhanced chemical and biological activity than graphite. Based on the existing number of graphene sheets the CNTs can be classified into two types Single Walled and Multiple Walled. CNTs comprising only one graphene sheet are termed as SWNTs, whereas CNTs with more than one graphene sheet are defined as MWNTs. SWCNTs have a diameter of a few nanometers, whereas MWCNTs have a diameter of several tens of nanometers. It is thousands of times smaller than the diameter of human hair and can be several hundred microns in length. The interatomic bond distance is reported to be 1.44 A0 between C-C atoms [6, 7]. Experimental and theoretical studies of CNTs have shown that they have an exceptionally high elastic modulus > 1 Tpa (diamond -1.2 TPa) and have a strength 10 to 100 times that of the strongest steel. In addition, CNTs exhibit remarkable thermal and electrical properties, including thermal stability up to 2800 °C in a vacuum, an electrical conductivity of approximately 103 Scm-1 with a carrying capacity several hundred times higher, and a thermal conductivity of approximately 1900 Wm-1K-1, which is two times higher than diamond [8, 9]. Since CNTs are known as potential electrode materials for devices with marked electrochemical applications due to their high surface area, and high electronic conductivity [10]. Furthermore, CNTs are ideal candidates for biosensing and electrochemical applications due to their ability to bind proteins without changing their activity [10–12]. CNTs reportedly show promising results in improving sensitivity, lowering detection limit, and reducing detection time and sample amount [13–15]. CNTs integrated with biological functionalities are anticipated to have enormous potential in future biomedical applications. This chapter outlines the recent advances and developments around biosensors which are being prepared with CNTs

CNT Applications in Biosensing  99 as the functional and prime sensing material. In the following section, we will overview and discuss various CNT-based biosensors.

5.2 CNTs-Based Biosensors A biosensor is a chemical sensing device that integrates the active biological components like nucleic acids, enzymes, antibodies, microorganisms, tissues, etc. The detection of the analyte is based on two steps: the recognition step and transducing step. In the recognition step, the analyte can be recognized either in the solution or in the atmosphere by the biological element. Further, because of the interaction between the recognition molecule and the analyte, a signal is transmitted to the detector. Signal transduction can take place electrochemically, optically, magnetically, calorimetrically, or gravimetrically, depending on the technique used. Biosensors could find applications in drug delivery, medical diagnosis health care and food safety, military applications, and environmental monitoring [16–18]. Among the above, healthcare application persists to be of key importance for biosensors. The first biosensor used to detect oxygen was developed in 1956. Since then, numerous different types of biosensors have been developed, along with the glucose biosensor for the analysis of clinical diabetes [18]. For biosensing applications, nanomaterials offer the possibility of a significant increase in sensitivity with a lower limit of detection. This improved performance is correlated with the nano-scale dimension of nanomaterials, which bestow them with a high surface-to-volume ratio. This high specific are aallowsimmobilization of large concentrations of bioreceptorunits compared to surface/volume biosensors. Moreover, many biomaterials may act as transducers due to their intrinsic physicochemical properties. During the past decade, CNT-based hybrid systems at the nano scale have been investigated vigorously for biological interfaces. As nanotubes are structured on the surface, their entire weight is centered on their surface layers. It is because of this property that tubules possess a particularly substantial unit surface which consequently decides their electrochemical and adsorption properties [19, 20]. Nanotubes possess a strong electronic response to molecules adsorbed on their surfaces, and their unprecedented unit surfaces make them a promising tool for the development of super miniaturized biological sensors. CNTs have been employed with a variety of biosensors, taking advantage of their useful physical and chemical properties. In general, depending on the type of transduction mechanism the biosensors can be classified as (i) electrochemical (ii) optical and (iii) field-effect sensors.

100  Functionalized CNT for Biomedical Applications

5.2.1 Electrochemical Biosensors Electrochemical sensors characterize the aged and frequently used class of biosensors, to date. They are popular due to their low-cost, relatively fast response times, ease of use, and portability. CNTs can be highly advantageous to electrochemical sensors because of their high surface areas and exceptional electrical properties. The sensitivity and detection limit of electrochemical sensors can be improved using CNTs; this can be achieved by increasing the active surface area and enhancing the immobilization of biological recognition elements of electrodes with improved electron transfer.

5.2.1.1 Electrochemical Enzyme Sensors An enzyme-coupled electrochemical biosensor induces an electric signal by catalyzing a reaction producing electroactive species. The biosensor usually consists of a reference electrode, a working electrode, and a counterelectrode. Analytes immobilized on the electrode which recognizes the target analyte catalyze electron transfer and contribute to the production of a voltage and current, depending on the enzymes used. Adsorption at the electrode surfaces is likely the easiest method for immobilization of enzymes on CNTs. The CNTs can be layered onto the electrodes either by drop-casting [21] or by direct synthesis of CNTs on the electrode surfaces [22]. Functionalization with enzymes onto electrodes CNTs in solution or directly onto electrodes is done via adsorption of enzymes or by covalently attaching enzymes to CNTs [23, 24]. More complex techniques involve dispersion of CNTs in ionic liquids [25] or in polymers such as Nafion [26], chitosan [27], or polymermediators [28]. Furthermore, researchers have also examined enzyme sensors using electrodes functionalized with CNTs and nanoparticles of gold [27] and platinum [29], and graphene. It has been reported that CNTs are typically randomly distributed over a polymer film or electrode surface in most CNT-based enzyme sensors. However, many examples of vertically aligned CNTs enzyme sensors have also been revealed. This illustrates an attractive technique where vertically aligned CNTs can be formed either by the direct synthesis [22, 31] or via accumulation of CNT dispersions onto functionalized substrates of Fe3+ precipitated hydroxides [31]. Further, enzymes can either be immobilized through adsorption [22] or by means of covalent bonding, generally through a -COOH functionalized CNT at the end group [30, 31]. It is noteworthy that CNTs have been explored for the achievement of direct

CNT Applications in Biosensing  101 electron transfer (DET) between electrode surfaces and redox enzymes. This is technically difficult to achieve due to the inside location of redox centers of enzymes as the protein shells are shielded inside. Using functionalized SWCNTs DET has been exhibited successfully by exploiting their nano-scale size and conductivity to make ‘molecular wires’ between redox cores of enzymes and the electrode surfaces [32, 33].

5.2.1.2 Electrochemical Immunosensors Immunosensor devices are based on the binding interactions between antigens and antibodies. In this approach, either the antibody or the corresponding antigen is immobilized on the sensor surface; a stable complex is formed via the binding of the corresponding antigen or antibody, which can be detected via various methods. Antibodies are frequently used in biosensing applications due to their highly specific bonding with corresponding antigens [34]. A commonly used method in CNT-immunosensors is sandwich assay, in this method, the antibodies bind at the electrode functionalized with CNTs, either directly or through another binding material present at the surface of the electrode. For detecting analyte protein binding, secondary antibodies with reporter molecules are used. The binding of reporter molecules to the immobilized analyte at the electrode surface will lead to a change in the electrical signal [35, 36]. Riberi and coworkers [36] designed an amperometry sensor (Figure 5.1) using a similar approach for detecting zearalenone (ZEA) with a detection limit of 0.15×10-12 g/mL. Zearalenone is a mycotoxin in maize. Here, MWCNTs were immobilized in a polyethyleneimine composite, over which deposition of AuNPs was done by drop-casting. The immunosensor

H2O2

H2O

+2e-

Carbon screen printed electrode Zeralalenona

Zearalenona labeled with horseradish peroxidase

Gold nanoparticles Multi-walled carbon nanotubes/Polyethyleneimine

Anti-ZEA poly-clonal antibody

Figure 5.1  Schematic representation of the electrochemical immunosensor to determine ZEA using the ZEA-pAb/AuNPs/MWCNT/PEI/CSPE [36].

102  Functionalized CNT for Biomedical Applications demonstrated outstanding analytical performance in terms of limit of detection (0.15 pg mL-1) and sensitivity (SC50 = 2 pg mL-1), as well as good repeatability and acceptable precision. Similarly, Rusling et al. [37] Reported vertically aligned CNTs-based Immunosensors. The group has developed SWCNT forests by dipping Nafion and Fe(OH)x functionalized graphite electrodes into dispersions of SWCNT. Further, HRP-labeled antibodies are used for carrying out the sandwich assay. Thus, here an amperometric immunosensor for matrix metalloproteinase-3 (MMP-3)was developed with a 4×10-12 g/mL detection limit. MMP-3was a biomarker related to squamous cell carcinoma. This group further used the same technique for detecting interleukin 6 (IL6) protein, a biomarker for oral cancer, with 0.5×10-12 g/mL detection limit. Additionally, Immunosensors using electrochemical impedance spectroscopy (EIS) eliminate the need for label molecules. Methods employing redox probes such as ferricyanide have also been reported [38]. In this method because of interaction between the sensor surface and the analyte, the process of electron transfer to the redox probe was retarded, causing a drop in current. By combining this method with square wave voltammetry, a detection limit of 0.163×10-12 g/mL was attained.

5.2.1.3 Electrochemical DNA Sensors Due to its distinctly specific hybridization, DNA has been found to be a fascinating biorecognition element for sensors. DNA sensors have promising significant applications including food safety, health and medicine, and antiterrorism. A general technique for immobilizing nucleic acids onto electrodes functionalized with CNT is to covalently attach the amine end of the nucleic acids to the electrode surface. Chen and co-worker [39] developed a DNA sensor based on CNTs that covalently bound aDNA capture probe with amine-terminus to a copper oxide nanowire dispersion and carboxylated SWCNTs. By employing this sensor, the anthrax lethal factor up to a limit of 3.5 was detected using differential pulse voltammetry (DPV). Similarly, using electrophoresis, Ghrera and co-workers [40] deposited carboxylated MWCNTs onto an indium tin oxide (ITO) electrode by covalently attaching oligonucleotides to the MWCNTs. The Figure 5.2(a-d) showed various steps involved in the fabrication of pDNA/MWCNT/ITO biochip. The biosensors covalently immobilized aminated capture probe has been discovered to precisely hybridize with its complementary DNA, allowing it to detect target oligonucleotide at concentrations as low as 1 fM with a response time of 1 minute. The device’s improved linear range and

CNT Applications in Biosensing  103 WE

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MWCNT/ITO electrode of chip EDC,NHS

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pDNA/MWCNT/ITO electrode of biochip

Figure 5.2  Stepwise illustration of microfluidic biochip fabrication for electrochemical detection of DNA hybridization. (a) Exfoliation of MWCNT via sonication with TOAB, (b) micropatterning of the ITO substrate and fabrication of microchannels, (c) EPD of MWCNT over the working electrode of the chip and (d) immobilization of DNA probe on the surface of MWCNT/ITO chip [40].

sensitivity can be attributed to better DNA loading thanks to MWCNT’s alteration of the electrode’s surface area. A linear association was found between the Rct values and the logarithm of the complementary DNA concentrations for both biochips. In the ten–decade concentration ranges from 1 fM to 1 M, a three-fold increase in Rct value was found for the pDNA/MWCNT/ITO biochip (Figure 5.3, curve  i). A considerable variance was detected with the pDNA/AS/ITO biochip (Figure 5.3, curve ii), showing that it is less suitable for sensing complementary target DNA. Furthermore, CNT-functionalized electrodes can also be used to immobilize nucleic acids utilizing gold/thiol interactions [41, 42]. Moreover, aptamers are potential substitutes for antibodies in biosensing applications. Since they have high stability, high specificity, and selectivity in binding and can be prepared cost-effectively [43, 44]. Similar sensors based on CNTs are also an active area of research [45]. Aside from detecting DNA, RNA, and protein, metal ion has also been detected by DNA sensors based on CNTs [46].

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Figure 5.3  Plot of the variation in Rct of (i) pDNA/MWCNT/ITO and (ii) pDNA/AS/ ITO biochips as a function of target DNA concentrations on logarithm scale. Error bars represent deviation of data points from linearity [40].

5.2.1.4 Non-Biomolecule-Based Electrochemical Sensors The CNT sensors comprising biomolecules are the most used electrochemical sensors, but there are numerous examples where biomolecules are not used as recognition elements in CNT-based electrochemical sensors. In the past few years, sensors based on molecularly imprinted polymers (MIPs) have been fascinating the researchers. MIPs are synthetically analogous to antibodies. Here, polymers comprising the target molecule are synthesized, bound to the polymer structure covalently or non-­covalently. This is followed by the separation of the target molecule from the polymer matrix, which in turn results in the formation of cavities (left behind the target molecule) of a certain shape, causing rebinding with the target molecule. Selective binding of MIP to the target in solution is like the binding of antibody/antigen. Compared to antibodies, MIPs may be more stable and inexpensive to mass-produce as recognition elements for sensors [47, 48]. For MIP applications, CNTs are potential candidates as they can enhance the electrical conductivity, mechanical properties, and porosity of polymer composites [49, 50]. There are many reports available, where MEPs containing functionalized MWCNTs were developed for the detection of the analyte involving epinephrine [49], myoglobin [51], and the cardiac biomarker troponin T [50]. These CNT-MIP sensors achieved detection at concentrations less than nanogram [49] or picogram [50].

CNT Applications in Biosensing  105 Also, CNTs have been extensively used with other nanomaterials to functionalize electrode surfaces for direct electrochemical detection of a variety of analytes, resulting in greater sensitivity. Along with CNTs, nanoparticles of gold and silver are also commonly used for functionalizing electrodes for detecting analytes like bisphenol A [52], urea [53] glucose [54], and volatile organic compounds (VOCs) [55]. Nanoparticles of graphene oxide and graphene are also usually employed along with CNTs to enhance the performance of electrodes [56, 57].

5.2.2 Optical CNT Sensors The optical biosensor detects analytes by measuring the light absorbed or emitted in catalytic and affinity reactions. Optical detection of the analyte can be achieved via absorption reflection, infrared, Raman, fluorescence, and resonance providing the nature of the biochemical reaction. The most popular optical detection techniques are fluorescence and surface plasmon resonance (SPR) because of their sensitivity and selectivity toward biochemical reactions. In fluorescent sensors and biosensors, CNTs are extensively used due to their fluorescent properties which are largely determined by their structure [58]. Further, CNTs can be functionalized with different types of biomolecules to utilize their fluorescent properties in the field of biosensing [59– 62]. Along with this, fluorescent quenching can also be accomplished with CNTs. Bio-recognition molecules like oligonucleotides can be labeled using Fluorophores. Once the oligonucleotides have been attached to the CNTs non-covalently, the fluorescent quenching of fluorophores can be done by the CNTs through Förster resonance energy transfer (FRET). When oligonucleotides are present with analyte molecules, the analytes bind preferably to them, displace the CNTs, and increase the fluorescent signal significantly [63–66]. Furthermore, SPR is one of the most sensitive optical techniques universally used for chemical and biological sensing. In this technique, exploitation of surface plasmon waves is done which formed between metal surface interface and dielectric, generated via optical stimulus. The detection of binding of molecules at the surface of the metal is done via alteration in the refractive index at the surface [67–69]. Lateral flow sensors based on CNTs have also been reported in the literature for visual detection. In this technique CNTs deposition at the test site leads to the formation of a black band when the antibody or oligonucleotide capture probe is bound to the test strip and used in combination with a CNT-labelled capture probe in a sandwich assay [70, 71].

106  Functionalized CNT for Biomedical Applications

5.2.3 Field-Effect CNTs Sensors Field-effect transistors (FETs) are microelectronic devices primarily used in electronic equipment and have three electrodes: a drain, a source, and an agate electrode. Typically, a reference electrode is used in conjunction with a liquid medium to replace the gate electrode for sensing applications. Charge carriers are influenced by changes in the electric field created by ionization or molecule binding at the surface of the channel, affecting the source/drain current. A variety of biorecognition elements have traditionally been applied to FETs in the field of biosensing. CNTbased field-effect biosensors have been a popular research field for many years because of their large surface area and high conductivity [72, 73]. Field-effect biosensors typically consist of single-walled CNTs, since SWCNTs are p-type semiconductors, making them perfect for forming semi-conductive channels [73]. There are two ways in which CNTs are dispersed in field-effect devices: either aligned between electrodes [74, 75] or arranged randomly between electrodes (the source and drain electrode) [76, 77]. To fabricate CNTs network devices, a dispersion of CNT sis made in a solvent like dimethylformamide (DMF), and further drop-casted onto the substrate. Alternatively, the electrodes can be directly fabricated on top of a substrate using standard photolithographic techniques, either between prefabricated electrodes or directly on the substrate [74]. CNTs-based FET biosensors have been widely applied to the detection of analytes based on nucleic acid and proteins [78, 79], employing various detect techniques, device configurations, and fabrication methods. These are also very frequently used in the field of immune sensors [80–82]. However, it is difficult to detect metal ions, biomolecules, and metabolites that are smaller in size, using standard FET-based detection methods. This limitation could be attributed to the fewer charged analyte, weak electric field effect due to small size, and few or single binding sites on transducer elements. These shortcomings also limit the signal amplification in sandwich assay methods. Using SWCNT-based FET biosensors [83, 84] these challenges have been overcome in many studies. This technique is based on the principle of competitive displacement, which involves biorecognition molecules bound to transducer immobilized analytes being displaced by unbound analytes present in the samples. FET-based CNTs biosensors are of great interest as new fabrication methodologies and materials provide cheaper, flexible, and mass-­producible potential devices [85, 86].

CNT Applications in Biosensing  107

5.2.4 CNT Human Strain Sensor The alkali siloxane-modified CNT showed excellent human body movement sensing, the organic molecules mimicked in CNT and conductivity showed at 157.64 S/m due to the high surface. And the sensing movement showed 40% sensing after 500 cycles of stretch in the leg. Further, the body joint stretching such as hand, fingers, wrists, legs elbows, Knees are analyzed sensing behavior as shown in Figure 5.4(a-c) achievement symbol of different direction movement, and the finger stretching increased the

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Figure 5.4  The fingers movement (a-c) and (d-f) leg knees stretching and their electrical signals [87].

108  Functionalized CNT for Biomedical Applications conductive is decreased because of stretching resistance increased. And Figure 5.4(d-f) showed that the electrical signal is changing with respect to the movement of the knee joint.

5.3 Conclusion CNTs offer a great platform to design a huge range of biosensors due to their distinct electronic properties like higher surface-to-volume ratio and outstanding mechanical properties. The major advantage of using CNTs in CNT-based biosensors is their enhanced detection performance of the target. Although, there are limited commercial applications of CNTs for sensing, and the reasons are the efficient growth of macroscopic size CNTs, the longevity of nanotube-based biosensors, the controllable growth of nanotubes on needed surfaces, the dispersion of CNTs in desired matrix or solution, and the control over their structure, and the scope of fabrication of biosensors using CNT. Along with this, the compatibility of biomaterial with the synthesis process must be checked. Since the synthesis of CNTs-based biosensors is done on an electrode surface or as an element of a field-effect device. For example, the requirement of high temperatures in the arc discharge and laser ablation synthesis methods or preparation of CNT scan harm many potential substrates. CNTs, therefore, account for universal and multifunctional nanostructures which imbibe the potential characteristics to be used in diagnostic and therapeutic applications. Although these may be employed as biosensors to detect cancer biomarkers with high sensitivity and cost-effective micro-fabrication, anticancer drugs could also be loaded on these and employed in oncology with therapeutic aims. They may also be applied for photoacoustic cancer cells molecular imaging and photothermic ablation of tumors.

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6 Applications of Functionalized Carbon Nanotubes in Drug Delivery Systems N. Palaniappan1, Małgorzata Kujawska2* and Kader Poturcu3 School of Chemicals Science, Central University of Gujarat, Gandhinagar, Gujarat, India 2 Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland 3 Department of Chemistry, Arts and Science Faculty, Suleyman Demirel University, Isparta, Çünür, Turkey 1

Abstract

An inimitable and the most prominent invention in the part of nanotechnology are carbon nanotubes (CNTs). High surface areas, high aspect ratios and nanosized stable tubes in CNTs unique structures, make them attractive materials in nanotechnology, nanomedicine, electronics, aerospace, biosensors, membranes, and capacitors applications. There are two main types of CNTs are single walled (SWNTs) and multi walled (MWNTs) carbon nanotubes. SWNTs and MWNTs consist of single long wrapped graphene sheet and multiple layers of graphite rolled over co-axially to form a tubular shape, respectively. Although one of the most important applications of CNTs is deliver drug molecules as nano containers, CNTs have disadvantages due to their hydrophobic nature which make these unique structures insoluble in organic and aqueous solvents. To overcome this challenge, synthetic functionalization process can be used. Thus, the solubility, biocompatibility enhances, and functionalized CNTs penetrate the cell and use as the potential vehicles for the drug delivery systems. This chapter covers the types, synthesis, toxic effects of CNTs and their applications in drug delivery to brain and cancer cells. Keywords:  Carbon nanotubes, drug delivery, nano particles, brain-targeted delivery, cancer

*Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (117–138) © 2023 Scrivener Publishing LLC

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118  Functionalized CNT for Biomedical Applications

6.1 Introduction In the last two decades, inorganic and organic nanostructures such as gold nanoparticles [1], SiO2 nanoparticles [2], dendrimers [3], liposomes [4], polymeric nanomaterials [5], magnetic nanoparticles [6] and carbonbased nanocontainers [7, 8] have played an important role for targeted drug delivery studies. Particular interest is given to the clusters of pristine and functionalized carbon nanotubes and their unique structures with interesting physical and chemical properties make them attractive in drug delivery applications [6, 9]. The last five years of developments on the “use of Carbon nanotubes in drug delivery” data collected using web of Science is given in Figure 6.1. CNTs, diamond and graphite are important allotropes of carbon atoms due to their valency. Among these allotropes, CNTs is considered the most attractive nanomaterials [10–12] due to their unique characteristics such as structure, dimension, strength, chemical stability, and electronic properties [13, 14]. CNTs, first observed in 1975 by Endo and later by S. Iijima in 1991, are basically formed of sp2-hybridized pure C atoms and belong to the fullerenes (C60) subfamily. CNTs have a cylindrical shape and are made of rolled-up graphene sheets [15–17]. Two different types of CNTs are: (a) single-walled carbon nanotubes (SWNTs), which are made up of a single graphene layer wrapped into a cylindrical structure, and (b) multiwalled carbon nanotubes (MWNTs), with a central tubule of nanometric diameter, surrounded by several graphitic layers spaced by a distance of about 0.34 nm (Figure 6.2) [18]. The properties can vary depending on

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CNTs in Drug Delivery Systems  119 (a)

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Figure 6.2  Aschematic diagram of (a) SWCNTs and (b) MWCNTs.

the types of CNTs [10, 19, 20]. SWNTs are one-dimensional nanomaterials with 1–2 nm diameters and 50nm–1cm lengths. On the other hand, MWNTs have much larger diameters (10–100 nm) [21]. Although CNTs can be synthesized naturally in a controlled flame environment by heating carbon black and graphites, produced nanotubes are mostly irregular in size, shape, mechanical strength, quality, and purity due to the uncontrollable natural environment [16, 22]. Nowadays, electric arc discharge (EAD), laser ablation technique (LA), and catalytic chemical vapor deposition (CVD) methods are widely used for synthesis of CNTs. In addition, high-pressure carbon monoxide disproportionation process (HiPCO) and plasma enhanced chemical vapored position (PE-CVD) [23] techniques are of recent interest [24]. These carbonaceous materials with their tubular shapes have novel applications in the fields of automotive industry, coatings and films, microelectronics, biotechnology, energy conversion and storage, medical and engineering applications [9]. Thanks to their specific atomic architecture coupled with high aspect ratio, characteristic configuration, excellent functionalization ability, and dynamic surface modification properties, CNTs are ideal nanomaterials for drug delivery applications [16]. In addition, CNTs with high inner volume and immobilization ability properties, are the most sought after nanomaterials in drug delivery [25] due to their versatile applications in controlled and targeted delivery, cancer treatment, bioavailability enhancement, transdermal delivery, theranostic applications, drug discovery, and delivery of proteins, peptides, RNA, DNA, siRNA [12, 16]. Generally, drug delivery systems can help the loaded drugs effectively enter the cells’ interior by endocytosis through the cell membrane.

120  Functionalized CNT for Biomedical Applications To deliver the drugs to the nucleus, the drug carrier must escape the endosomal compartment and releases loaded drug in cytosolic compartments [26, 27]. CNTs are chosen widely for their high loading of cargo molecules and excellent cell penetration ability, which enables them to deliver drug substances into neoplastic cells for selective destruction and to reduce the distribution of drugs to normal adjacent cells that aid in avoiding the toxicity of the cells, mainly in cancerous cells as chemotherapeutic and imaging agents [27]. Therefore, these nanotube systems can solve transport problems for pharmacologically relevant compounds [28, 29]. In the literature, Maleki and Soltanabadi investigated the adsorption capability of the pristine and functionalized carbon nanotubes of anticancer drug dacarbazine. They found that despite the adsorption of dacarbazine molecules on the outer sidewall of the carbon nanotubes, the spontaneous encapsulation of drug molecules into the cavity of carbon nanotubes is observed [30]. Bhatnagar et al. develop a new drug carrier system with SWNTs to carry cancer drug gliotoxin. They found that effective drug release was monitored against cervical and breast cancer cells [10]. Kamel et al. investigated the solvent and co-solvent effects on the adsorption strength between flutamide used for prostate cancer treatment and SWNTs for different configurations in the water solution based on the density calculation. The negative values of the adsorption and solvation energies demonstrate that all theoretical calculations are stable, and the interaction of the drug molecule with the nanotube sidewall is a spontaneous process [31]. Arora et al. evaluated cellular uptake of anticancer drug docetaxel conjugated MWNTs in human breast cancer cells (MCF-7 and MDAmb-231) and they reported that drug conjugated nanotube systems is effective against cancer cells [32]. Saberinasab et  al. investigated the efficiency of polyethylene glycol functionalized carbon nanotubes to deliver anticancer drug temozolomide based on the density functional study [33]. Sobhani et al. synthesized poly citric acid functionalized MWNTs and then potent anticancer agent paclitaxel was conjugated to the carboxyl functional groups of poly citric acid. This system was suitable for the release of the drug in tumor tissues and tumor cells [34]. Anbarasan et al. formulated doxorubicin-loaded f-MWNTs to deliver this drug-­nanotube system only to cancer cells by using pH difference. This formulated system have great potential as a drug carrier [35]. Although CNTs which are widely used in biomedical applications must be biocompatible, soluble, and non-toxic, synthesized CNTs have highly hydrophobic surfaces that make their unique structures insoluble in organic solvents, aqueous system and particularly in biologically relevant buffers [27, 36, 37]. Due to the strong van der Waals forces, these structures highly agglomerated. Therefore, obtaining a well-dispersed suspension of

CNTs in Drug Delivery Systems  121 CNTs in water or organic solvents without physical or chemical modifications is challenging [38, 39], and surface chemistry modifications or functionalization of CNTs is required to modify them with a wide variety of molecules [36]. There are several approaches for the functionalization of CNTs, including covalent functionalization and non-covalent coating of CNTs [37, 40]. Within the covalent strategy, the organic functionalization of SWNTs and MWNTs by 1,3-dipolar cycloaddition of azomethineylides is a powerful methodology for generating amino-functionalized carbon nanotubes with high water-soluble property [41]. The ‘ends and defects’ and ‘side walls’ functionalization [42] are two subcategories of covalent functionalization. The oxidation and cycloaddition reactions are the most common types of covalent functionalization of CNTs [18, 43–46]. The oxidation reactions is carried out with strong acid such as nitric acid [46], and various functional groups can be attached to CNTs via [42], arylatio [47], and diazonium salt chemistry [48–50] reactions. During the oxidation process, -COOH groups are formed at the ends of carbon nanotubes and the defects on the sidewalls. SWNTs are broken to very short lengths (100– 300 nm) [20, 51]. Although oxidized CNTs have water soluble properties, they could not use for biological applications due to the high salt content of most biological solutions, which cause aggregation of CNTs. To overcome this limitation, further modification is performed with the attachment of hydrophilic polymers (PEG) to oxidized CNTs, yielding CNT-polymer conjugates stable in biological environments [21]. Non-covalent functionalized nanotubes do not compromise the physical properties of CNTs, but improve solubility and processability. Non-covalent functionalization mainly involves surfactants, biomacromolecules, or wrapping with polymers. CNTs can be well dispersed in water using anionic, cationic, and non-ionic surfactants [52, 53]. Sodium dodecyl sulfate (SDS) [54, 55] and sodium dodecylbenzene sulfonate (NaDDBS) are commonly preferred anionic surfactants to decrease CNT aggregation in water. Cetrimonium bromide (CTAB), 2-(methacryloyloxy)ethyl]trimethylammonium chloride (MATMAC) and Triton X-100, Triton X-305 are widely used cationic and non-ionic surfactants, respectively [20]. Nowadays, nanotechnology is revolutionizing the approaches to different fields from manufacture to health.

6.2 Nanoparticles-Doped Carbon Nanotubes The surface functionalizing of the CNTs is a well-established approach to improving CNTs solubility and tuning the drug uptake properties of CNTs

122  Functionalized CNT for Biomedical Applications [56, 57]. The most common two functionalization methods are: electrical doping and chemical doping. Electrical doping is a physical doping method that preserves the lattice structure or chemical composition of the graphene sheets, whereas chemical doping involves substitutional atoms (e.g., N, B, S, Si) [58–61] or adsorbed inorganic (e.g., NO2, HNO3) [62–64] and organic molecules (e.g., toluene) [65]. Substitution (doping) of carbon atoms of CNTs with B or N is the most effective way to alter the electronic structure and chemical reactivity of CNT because additional electronic states around the Fermi level are introduced (Figure 6.3) [66]. In biomedical applications, Nitrogen-doped CNT in rats showed low toxicity and better biocompatibility [67, 68]. Nitrogen-doped carbon nanotube (N-CNSs) sponges have good electroactive, superhydrophobic, and oil-absorbent properties, as well as good absorption capacities with a loading capacity of up to 100 times its own weight [8]. The doping of CNTs with all of these characteristics make these structures appropriate carriers for various chemotherapeutic agents for local delivery, mainly for solid tumors in which the side-effects induced by chemotherapeutics significantly reduce the quality of life of patients [69]. The B atom functions as a site for adsorption of aromatic molecules, which therapeutic molecules often are, on common anticancer drug doxorubicin [68]. Therefore, both N- and B-doped nanotubes have potential biomedical uses [70, 71].

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Figure 6.3  Doped CNTs for drug delivery system [66].

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CNTs in Drug Delivery Systems  123

6.3 Brain-Targeted Delivery The delivery of therapeutics to the central nervous system (CNS) is one of the major obstacles in treating neurological diseases. The significant drug development challenges are mainly related to crossing the blood-brain and blood-cerebrospinal fluid (CSF) barriers and systemic effects, including binding with plasma proteins, biotransformation, and excretion. The blood-brain barrier (BBB) is formed by endothelial cells, astrocytes, and pericytes. Tight junctions between endothelial cells prevent the entering of about 98% of all small molecules, and approximately 100% of large molecules cannot reach the neural tissue. The blood-CSF is a selective filter separating the blood circulation from the cerebrospinal fluid and controlling molecules’ entrance into the CNS. Since this barrier is about 1000times smaller than the BBB, it is less relevant regarding CNS drug delivery [72, 73]. CNTs with outstanding cell-penetrating ability offer the potential to be developed as carriers to deliver CNS therapeutic molecules [72]. Their needle-like shape makes them suitable to cross the BBB without causing any apparent cell damage [26]. Kafa et al. have shown that in vitro “wide” MWCNTs (∼35.9 nm diameter) passed a BBB co-culture model in a higher percentage than the “thin” MWCNTs (∼9.2 nm diameter). In vivo, brain uptake of MWNTs with smaller diameters was favored after systemic injection. Conjugation of CNTs with angiopeptin-1 (ANG) improved brain distribution of wider MWCNTs [74]. Of note, CNTs can reach the CNS through the systemic, olfactory, and trigeminal pathways, which can be used as administration routes [75]. As they have the capability of delivering a vast range of cargos, including drugs, antigens, genetic materials, and biological macromolecules via the BBB [76]. CNTs represent the potential of innovative nano platforms for medicine delivery in the treatment of various neurological conditions, including infections, brain, and spinal trauma, stroke and brain cancer that has been supported in several in vitro and in vivo studies [75]. Ren et al. have developed the drug delivery system by modifying PEGylated oxidized MWCNTs with angiopep-2 (ANG) to target LRP receptors on BBB and glioma cells, which successfully delivered anticancer doxorubicin for glioma treatment in a mouse model. Notably, nanomedicine showed lowered cardiotoxicity compared to free doxorubicin [77]. You et al. have delivered oxaliplatin using carboxylated MWCNTs dual-functionalized with cell-penetrating peptidetrans-activating transcriptional activators–Polyethylenimine biotincopolymer and oxaliplatin [78]. Moreover, perspectives in neuroncology are also related to the optical feature of the CNTs, which absorb near-infrared radiation (NIR) not

124  Functionalized CNT for Biomedical Applications adsorbed by tissues. Thanks to targeting ligands, the adsorbed radiation can be converted to heat in a time and dose-dependent manner to control exclusively in the cancer cells temperature increase, i.e., hyperthermia triggering apoptosis and other death pathways [79]. In developing Alzheimer’s disease (AD), acetylcholine (Ach) deficiency appears, leading to memory and cognition impairment. Due to its hydrophilic nature, Ach does not cross the BBB; therefore, its noncovalent loading into SWCNTs to distribute this neuromediator to the brain has been developed [80]. The development of berberine, an isoquinoline alkaloid used in dementia, loaded multi walled CNTs coated with polysorbate and phospholipid, has been found to be effective in managing AD by Lohan et al. [81]. Costa et al. have demonstrated that oxidized MWCNTs improved the accumulation of Pittsburgh Compound B derivatives, amyloid-β-binding molecules with low BBB permeability, in mice brains after intravenous injection [82]. Importantly, Li et al., based on molecular dynamics simulation results, have suggested the ability of CNTs for direct inhibition of Aβ(16-22) and full-length Aβ fibrillation in AD [83]. Moreover, functionalized SWCNTs have been demonstrated to restore normal autophagy by reversing abnormal activation of mTOR signaling and deficits in lysosomal proteolysis, thereby improving the elimination of the mutant amyloid protein [84]. Carboxylated SWCNTs have been developed to sustain release of nanocarriers for delivering levodopa concerning Parkinson’s disease (PD) [85]. Guo et al. loaded functionalized carboxylated SWCNT-PEG-lactoferrin with high amounts of dopamine for its targeting delivery into the striatum of parkinsonian mice after intraperitoneal injection [86]. Brain-targeted delivery of β2AR agonists downregulating α-synuclein gene expression using functionalized SWCNT or MWCNTs systems has been reviewed to overcome peripheral off-target side effects of the β2AR agonists offering potential use in managing PD [87]. Lee et  al. have demonstrated the neuroprotective effects of amine-modified SWCNTs (a-SWNTs) in a rat model of stroke. Low levels of apoptotic, angiogenic, and inflammation markers reflected the effective blockage of ischemic damage by a-SWNTs [88]. Additionally, CNTs impregnated with subventricular zoneneural progenitor cells have been reported to repair damaged neural tissue following a stroke in rats reducing volume and area of infarct cyst that was accompanied by improved behavior [89]. It is projected that CNTs could be developed for therapeutic application in nerve regeneration. CNTs adapted to coating with nerve growth factors and neural stem cells accelerated the growth of neural networks. MWCNTs coupled with electrical stimulation can exert a synergistic effect on promoting neurite outgrowth. Although those great efforts have been made, only a

CNTs in Drug Delivery Systems  125 few studies in vivo on CNTs applications aimed at accelerating the growth of neural networks have been reported so far [90]. Regarding the biocompatibility of CNTs, the potential induction of oxidative stress and related oxidative damaged products accumulation, DNA destruction, and inflammatory response are significant obstacles for their applications in neurological diseases therapy. Moreover, brain degradation and excretion are substantial concerns, a double-edged sword. Degraded and excreted too rapidly of CNTs may affect the therapeutic effect, while too high retention may cause brain damage. The rate of degradation and excretion of CNTs can be, however, tailored to the intended pharmacokinetics parameters. Moreover, contamination with byproducts of the synthesis, mainly metals and carbonaceous particles, decreasing cell viability, causing lipid peroxidation and oxygen radical formation are often responsible for the CNT toxicity. Therefore, only well-tailored, highly purified, and suitably coated CNTs can provide an acceptable safety level [75, 90].

6.4 The Organic Molecules Functionalized CNTs as Drug Delivery Vehicles 17-βEstradiol decorated MWNTs studies are performed with Doxorubicin (DOX) as drug delivery for breast cancer model. The results are showed that the DOX model has excellent inhibition efficiency in in vitro and in vivo environments. They observed that DOX along with E2- PEGMWCNT showed that specifically targeted only nuclear by estrogen receptor through due to sudden environmental changes and other genetic problems [91–95]. But still does not fulfill the drug delivery system poor materials also vary the materials physicals and chemicals properties. Hence traditional drugs delivery inorganic/organic materials failed high efficiency due to lack of chemicals properties [96–98]. In the past decades, pharmacological are used nanomaterials synthesis and delivery efficiency. As shown in Figure 6.4, nano stable store in the open-walled nanotubes along with active pharmacy materials. Nanotubes may increase functional materials released. With the varied size of nanoparticles the pharmacokinetics increased, and toxicity also decreased due to chemical properties change in molecular levels [99, 100]. However, the as shown in Figure 6.4b showed that nitrogen nonbonding electrons play a key role in transporting the active drug molecules by the support of electron exchange in the carbon nanotube moiety [101–110]. Hence the nitrogen lone pair electron could support the stable ambient environment and rapidly deliver the active drug molecules.

126  Functionalized CNT for Biomedical Applications N– N+

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Figure 6.4  (a) Nanoparticles doped CNT as drug carrier (b) Nitrogen-based active molecules functionalized CNT.

6.5 Functionalized CNTs with Nanoparticles for Drug Active Molecular Mechanism The CNTs have plenty of none bonding electrons in the structure. As shown in Figure 6.5, the nanoparticles as well as functionalized hetero organic molecules could be able to access the foreign particles and molecules in the microstructure [111–115]. We suggested that drug active molecules successfully release.

6.5.1 Future of Scope of Functionalized Carbon Nanotube Drug Delivery Application Carbon based nano materials are vastly abundant in earth crust and make it new derivatives is low cost. We are proposed these functionalized carbon nano materials significantly promote the drug delivery bio science [116–119]. The carbon nanotube based organic and inorganic materials are improving the targeted delivery system by supportive high surface area and pore volume of carbon materials [120–125]. Hence carbon materials will derive several new molecules for drug deliver system by the modification SP2 carbon structure [126–133]. However the effective carbon based molecules challenge by selective targeted molecules synthesis.

CNTs in Drug Delivery Systems  127 OH

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Figure 6.5  The schematic diagram for drug-loaded carbon nanoparticles releasing on respective active places.

6.6 Conclusion We have discussed different drug delivery nano particles incorporated carbon nanotube and organic molecules functionalized materials. The nano particles decorated carbon nanotube showed improve drug deliver but toxicity issued there. Hence the biologically active organic compound showed excellent delivery efficiency the organic molecules modified carbon ­nanotubes will be significant drug delivery vehicle in future.

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128  Functionalized CNT for Biomedical Applications 2. Li, Y., Li, N., Pan, W., Yu, Z., Yang, L., Tang, B., Hollow mesoporous silica nanoparticles with tunable structures for controlled drug delivery. ACS Appl. Mater. Interfaces, 9, 3, 2123–2129, 2017. 3. Chauhan, A.S., Dendrimers for drug delivery. Molecules, 23, 4, 938–945, 2018. 4. Pattni, B.S., Chupin, V.V., Torchilin, V.P., New developments in liposomal drug delivery. Chem. Rev., 115, 19, 10938–10966, 2015. 5. Marasini, N., Haque, S., Kaminskas, L.M., Polymer-drug conjugates as inhalable drug delivery systems: A review. Curr. Opin. Colloid Interface Sci., 31, 18–29, 2017. 6. Rezaei, A., Morsali, A., Bozorgmehr, M.R., Nasrabadi, M., Quantum chemical analysis of 5-aminolevulinic acid anticancer drug delivery systems: Carbon nanotube, –COOH functionalized carbon nanotube and iron oxide nanoparticle. J. Mol. Liq., 340, 117182, 2021. 7. Kaur, J., Gill, G.S., Jeet, K., Applications of carbon nanotubes in drug delivery: A comprehensive review. A comprehensive review, in: Characterization and Biology of Nanomaterials for Drug Delivery: Nanoscience and Nanotechnology in Drug Delivery, pp. 113–135, Elsevier, 2018. 8. Karimzadeh, S., Safaei, B., Jen, T.C., Prediction effect of ethanol molecules on doxorubicin drug delivery using single-walled carbon nanotube carrier through POPC cell membrane. J. Mol. Liq., 330, 115698, 2021. 9. De Volder, M.F.L., Tawfick, S.H., Baughman, R.H., Hart, A.J., Carbon nanotubes: Present and future commercial applications. Science, 339, 6119, 535– 539, 2013. 10. Bhatnagar, I., Venkatesan, J., Kim, S.K., Polymer functionalized single walled carbon nanotubes mediated drug delivery of gliotoxin in cancer cells. J. Biomed. Nanotechnol., 10, 1, 120–130, 2014. 11. Lu, X. and Chen, Z., Curved Pi-conjugation, aromaticity, and the related chemistry of small fullerenes ( (b) Proton MR images of an injected rat: (i) before, (ii) 6 h after, and (iii) 7 days after injection showing signal variation in the spleen and kidneys. The superimposed signal intensity color maps over kidneys and spleen indicate the MR signal intensity drops post injection. The American Chemical Society owns the rights to this work [104].

212  Functionalized CNT for Biomedical Applications

9.2.5 Nuclear Imaging Outside markings, such as radioisotopes, can also be used to increase the versatility of SWNT-based imaging studies, in addition to embracing the natural properties of SWNTs. Wang et al. discovered that the 125I may be used to track the bio distribution of SWNTs in living things [105]. The same group that had been following the long-term bio distribution of MWNTs had also figured out a 14C-based alternative to 125I [106]. By labelling CNTs with 111In, the methodology of tiny single-photon Emission computerized scanning tomography (miniature SPECT) was successfully acquired [107]. As a result, McDevitt and colleagues used 86Y to label SWNTs for Positron Emission Tomography (PET) imaging [108]. Several studies have used radionuclide-labelled SWNTs for in vivo cancer imaging in mice models [98, 99]. Through a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic corrosive (DOTA) chelator, Liu et al. [109] used 64Cu to label PEGylated SWNTs with RGD peptide synthesis in 2007. The specific restriction between RGD peptide and integrin av3 overexpressed on U87MG growth cells as well as growth vasculatures was recognized in vivo growth designated PET imaging of U87MG tumors using 64Cu/ DOTA-named SWNT-PEG-RGD. In addition to the RGD peptide, hostile to CD20 antibodies were generated using 111In-named SWNTs and utilized to target human Burkitt lymphoma [100]. Radioisotopes might be incorporated into nanotubes for radiolabeling instead of using traditional chelation technology to obtain radiolabeled CNTs [101]. A 125I-marked SWNT can be imaged using SPECT/CT after Na125I is inserted into the vacant design of SWNTs, according to Hong et al. The advantages of radio-marked SWNTs over other methods are that they have no depth limitation in tissue infiltration and are highly responsive. The use of CNTs for nuclear imaging has slowed down considerably in the last couple of years, with little significant progress, according to our best knowledge. Overall, combining nuclear imaging with other imaging processes might be extremely beneficial to the advancement of CNT-based bio-imaging assays.

9.3 Prospects and Challenges Different imaging methods for SWNTs have their own set of advantages and disadvantages. Because of the low native tissue dissipation and much-reduced photon scattering in the NIR-II district compared to the

CNT for Biomedical Imaging  213 apparent (400-700 nm) and traditional NIR-I region, fluorescence imaging in the NIR-II natural, straightforward window with SWNTs enables in vivo optical imaging with profound tissue entrance and high spatial goal (700900 nm). Despite this, the QY of SWNTs is still insufficient. The goal of this project is to develop new approaches to NIR-II imaging using SWNTs with multi-shaded fluorescence, which will require the development of simple union and filtration techniques to get SWNTs with pure chirality. Solid resonance Raman scattering is seen in SWNTs, which have a large dissipating cross-segment. Raman imaging using SWNT-tests has a high sensitivity and is unaffected by image fading and quenching. Noble metal-covered SWNTs display also enhanced Raman signals as a result of the SERS effect, allowing for much faster Raman planning. In any case, even with the SERS augmentation, acquiring a Raman picture using standard Raman planning takes roughly 10 minutes. SWNT-based Raman imaging may become a feasible technique for genuine natural exploration as technology improves. In addition, mass-based Raman signals of SWNTs will be greatly enhanced when chirality-cleaned SWNTs are used with the majority of nanotubes. Other CNT-based imaging approaches have shown considerable potential in biological imaging, such as PA imaging, MR imaging, and atomic imaging. Coordination of many imaging modalities might boost each imaging technology’s strengths while overcoming its inherent limits. Carbon nanotubes’ multifunctionality is the actual benefit of employing them as an imaging test. As opposed to many other nanomaterials used in bioimaging, CNTs possess increased optical and appealing properties, which eliminate the need to design complex nanostructures in order to accomplish diverse capabilities. Furthermore, the advantages of CNTs (such as medication and quality delivery, photograph warm therapy) [19, 76] combined with imaging would make them excellent theranostic Nano-stages. A wide range of animal diseases has been linked to CNTs without adequate surface functionalization. In addition to the limitations of each approach, one important test for the therapeutic use of CNTs is their longterm toxicity [102, 103]. CNTs without adequate surface functionalization have been known to cause poisoning in vivo [104–106]. Researchers have found that CNTs with well-planned surface coatings, such as PEGylation, are not toxic in vitro or in vivo [51, 79, 107–111] and are excreted from animals via renal or perhaps facial pathways [112–115]. In a number of studies, CNTs have been claimed to degrade when chemically oxidized [116, 117]. In any event, it is understandable that, while CNT-based biomedical

214  Functionalized CNT for Biomedical Applications imaging has enormous promise in basic biomedical research as well as in vitro/ex vivo clinical diagnosis, there will be significant challenges in using these nanomaterials in real-world clinical applications.

9.4 Conclusion Several modalities of biomedical imaging have been outlined in this chapter. They offer versatile means of tracking, detecting, and imaging biological systems due to their intrinsic physical properties including resonance Raman scattering, NIR photoluminescence, and strong NIR optical absorption, along with various external organic dyes, radionucleotides, and magnetic labels. However, CNT-based biomedical imaging still has many challenges to overcome before it can be widely used for clinical imaging, but these nanomaterials hold great promise in the biomedical field, and may promise novel tools for molecular imaging in biological systems.

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CNT for Biomedical Imaging  221 84. Yang, K., Hu, L., Ma, X., Ye, S., Cheng, L., Shi, X., Li, C., Li, Y., Liu, Z., Multimodal imaging guided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles. Adv. Mater., 24, 1868–1872, 2012. 85. Ku, G., Zhou, M., Song, S., Huang, Q., Hazle, J., Li, C., Copper sulfide nanoparticles asa new class of photoacoustic contrast agent for deep tissue imaging at 1064nm. ACS Nano, 6, 7489–7496, 2012. 86. Agarwal, A., Huang, S., O’Donnell, M., Day, K., Day, M., Kotov, N., Ashkenazi, S., Targeted gold nanorod contrast agent for prostate cancer detection by photoacoustic imaging. J. Appl. Phys., 102, 064701–064704, 2007. 87. Robinson, J.T., Welsher, K., Tabakman, S.M., Sherlock, S.P., Wang, H., Luong, R., Dai, H., High performance in vivo near-IR(N1μm) imaging and photothermal cancer therapy with carbon nanotubes. Nano Res., 3, 779–793, 2010. 88. Liu, X., Tao, H., Yang, K., Zhang, S., Lee, S.-T., Liu, Z., Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors. Biomaterials, 32, 144–151, 2011. 89. Moon, H.K., Lee, S.H., Choi, H.C., In vivo near-infrared mediated tumor destruction by photo thermal effect of carbon nanotubes. ACS Nano, 3, 3707–3713, 2009. 90. Avti, P.K., Hu, S., Favazza, C., Mikos, A.G., Jansen, J.A., Shroyer, K.R., Wang, L.V., Sitharaman, B., Detection, mapping, and quantification of single-walled carbon nanotubes in histological specimens with photoacoustic microscopy. PloS One, 7, e35064, 2012. 91. Galanzha, E.I., Shashkov, E.V., Kelly, T., Kim, J.-W., Yang, L., Zharov, V.P., In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells. Nat. Nanotechnol., 4, 855–860, 2009. 92. de la Zerda, A., Bodapati, S., Teed, R., May, S.Y., Tabakman, S.M., Liu, Z., Khuri-Yakub, B.T., Chen, X., Dai, H., Gambhir, S.S., Family of enhanced photoacoustic imaging agents for high-sensitivity and multiplexing studies in living mice. ACS Nano, 6, 4694–4701, 2012. 93. Merbach, A.E. and Tóth, É., The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, Wiley, Chichester (W. Sx.) etc, 2001. 94. Sitharaman, B., Kissell, K.R., Hartman, K.B., Tran, L.A., Baikalov, A., Rusakova, I., Sun, Y., Khant, H.A., Ludtke, S.J., Chiu, W., Super paramagnetic gadonanotubes are high performance MRI contrast agents. Chem. Commun., 3915–3917, 2005. 95. Al Faraj, A., Cieslar, K., Lacroix, G., Gaillard, S., Canot-Soulas, E., Cremillieux, Y., In vivo imaging of carbon nanotube biodistribution using magnetic resonance imaging. Nano Lett., 9, 1023–1027, 2009. 96. Al Faraj, A., Fauvelle, F., Luciani, N., Lacroix, G., Levy, M., Cremillieux, Y., CanetSoulas, E., In vivo biodistribution and biological impact of injected carbon nanotubes using magnetic resonance techniques. Int. J. Nanomedicine, 6, 351–361, 2011.

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10 Functionalized Carbon Nanotubes for Artificial Bone Tissue Engineering Sougata Ghosh1 and Ebrahim Mostafavi2,3* Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India 2 Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA 3 Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA 1

Abstract

Bone-related defects such as arthralgia, congenital malformation, osteoporosis, sports injuries, tumors, and traumatic accidents are often associated with insufficient self-repair and require additional medical intervention. Conventional orthopedic therapeutic approaches are occasionally challenged by non-integration, graft rejection, and implant failure. Advances in the area of nanotechnology have provided complementary and alternative approaches to ensure regeneration of the bone tissues by induction of osteogenesis followed by osseointegration. This chapter highlights the promises of carbon nanotubes (CNTs) which are considered as potential nanostructures for developing scaffolds for bone tissue engineering. The high surface area-to-volume ratio and superior electronic, mechanical, and thermal properties of CNTs provide an ideal surface for multifunctionalization of an array of biomolecules that include hydroxyapatite, polycaprolactone, polymethyl-­ methacrylate, poly(lactide-co-glycolide), poly-L-lactic acid, polyvinyl alcohol, poly(ether ether ketone)-calcium polyphosphate cements, chitosan, collagen, bacterial cellulose, and silk fibroin. Further, strategies like solution-based processing, polymerization, melt-based processing, and grafting methods are used to fabricate CNTs based 2D/3D constructs that may combine cells, growth factors, and scaffolds as key elements in regenerative medicine. Similarly, CNTs based 3D-printing methods can result in desired microscale features or electrochemical properties to facilitate robust cell adhesion, proliferation, and differentiation. However, further *Corresponding author: [email protected]; [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (225–256) © 2023 Scrivener Publishing LLC

225

226  Functionalized CNT for Biomedical Applications investigation is required to understand the exact mechanisms behind osteoinductive effects of CNTs based nanocomposites and associated toxicity before it comes to practice. In view of the background, it is evident that CNTs are critical for developing regenerative medicine and bone tissue engineering that can significantly revolutionize future healthcare. Keywords:  Carbon nanotubes, scaffolds, bone tissue regeneration, functionalization, osteogenic induction, osseointegration

10.1 Introduction Bone defects affecting millions of people globally each year may be associated with traumatic accidents, sports injuries, arthralgia, genetic disorders, osteoporosis, and cancer. Bone tissue regeneration is challenged by critical-sized defects and deeper wounds which occasionally require medical interventions such as surgical procedures for grafting and implantation. The autografts, allografts, and xenografts are the most accepted treatment method for bone repair, where autograft facilitates efficient osteogenesis and osteoconduction without any immunogenicity [1, 2]. Several complications in this surgical process include chronic pain, bleeding, and injury to the nerves. Moreover, lack of donors and longer duration for the surgery are considered as major limitations [3, 4]. On the other hand, although allografts can induce osteogenesis and possess osteoconductive properties, graft rejection, contraction of transmissible diseases, and infections are some of the main drawbacks [5, 6]. Further, the mechanical properties of allografts are not identical to the load-bearing bone [7]. Alternative strategies for restoring, repairing, and replacing bone tissues using engineered biomaterials have recently gained more popularity. Nanotechnology is employed to develop scaffolds for delivery of cells, peptides, oligonucleotides, and drugs [8, 9]. Several biomaterials composed of metal, metal oxide, mesoporous silica, and polymeric nanostructures are used widely in tissue engineering [10, 11]. Carbon nanotubes (CNTs), which are rolled into single or multiple graphene sheets termed singled-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs), respectively, are extensively explored for bone tissue engineering. The development in the area of CNTs based composite materials in the past two decades is depicted in Figure 10.1. These materials have exotic physico-chemical and opto-electronic properties that are notably distinct from the other allotropes of carbon, such as graphite, diamond, and fullerenes [12].

CNTs for Bone Tissue Engineering  227 (A) fullerene Carbon

SWCNTs

1D

0D

2D

3D

nano diamond graphene

diamond

Carbon dots

MWCNTs

graphite

osteob osteog last bone reenic differenti bone ti generation ation ssue en gineerin g

300

Number of articles published

GO

als teri ma thods n e t i o pos n me luati s Com ricatio ty eva ation c Fab roper appli p e Bio vativ o Inn

250

le stab ng ally ly stro tible c i m l a Che hanica comp ssible y c l e l e c a c M ic lly a log Bio omica n Eco

(B)

200 150 100 50 0 carbon (B1) graphene (B2)

diamond (B3)

200 0−N ow befo re 2 000

Figure 10.1  (A) Representative carbon-based materials: 3D graphite and diamond; 2D graphene and GO; 1D CNTs; 0D nano diamond, fullerene, and C-dots; (B) Number of articles published on the applications of carbon-based materials in BTE-related studies, search results obtained by Google Scholar on September 17, 2019. (B1) Blue column represents article number based on search results with the exact phrase “carbon” and “osteoblast” occurring in the title of a paper; the green column represents article number based on searching results with the exact phrase “carbon” and “osteogenic differentiation” occurring in the title of a paper; red column represents article number based on searching results with the exact phrase “carbon” and “bone regeneration” occurring in the title of a paper; black column represents article number based on searching results with the exact phrase “carbon” and “bone tissue engineering” occurring in the title of a paper. Columns of B2 (graphene), and B3 (diamond) are defined similarly as in the case for the column of B1 (carbon). Reprinted with permission from Peng Z, Zhao T, Zhou Y, Li S, Li J, Leblanc RM (2020) Bone tissue engineering via carbon-based nanomaterials. Adv. Healthcare Mater. 9(5), 1901495. Copyright © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [16].

CNTs are mainly fabricated using arc discharge, chemical vapor deposition (CVD), and laser ablation methods followed by purification to remove the metal impurities from the catalysts used during the synthesis process. CNTs are significant materials for reinforcement, drug delivery [13], sensing, and bioimaging, hence considered as attractive theranostic agents [14, 15].

228  Functionalized CNT for Biomedical Applications Table 10.1  CNTs based composite for bone tissue engineering. CNTs

Composite material

Functionalized ligand

Cells/models

References

MWCNTs

hydroxyapatite

-

hBMSC

[22]

MWCNTs

hydroxyapatite

graphene oxide

MDCK cells

[23]

MWCNTs

hydroxyapatite

-

L929 mouse fibroblast cells

[24]

MWCNTs

hydroxyapatite

stainless steel surface

MG-63 human osteoblast cells

[25]

MWCNTs

hydroxyapatite

gellan, amylopectin

MG-63 human osteoblast cells

[26]

MWCNTs

poly(εcaprolactone)

-

human adipose derived stem cells (hADSCs)

[27]

MWCNTs

poly(εcaprolactone)

-

MG-63 human osteoblast cells

[29]

MWCNTs

poly(εcaprolactone)

-

rat bone-marrowderived stromal cells (BMSCs)

[30]

poly(εAminecaprolactone) functionalized single-walled carbon nanotubes (aSWNTs)

-

rat bone marrowderived mesenchymal stem cells (rBMSCs)

[31]

MWCNTs

poly(εcaprolactone)

nano MG-63 human hydroxyapatite osteoblast cells

[32]

MWCNTs

polymethylmethacrylate (PMMA)

chitosan

MG-63 human osteoblast cells

[33]

CNTs

polymethylmethacrylate (PMMA)

hardystonite

MG-63 human osteoblast cells

[34]

CNTs

Poly(lactide-coglycolide) (PLGA)

-

MC3T3-E1 osteoblasts

[35]

(Continued)

CNTs for Bone Tissue Engineering  229 Table 10.1  CNTs based composite for bone tissue engineering. (Continued) Composite material

Functionalized ligand

Poly(lactide-coglycolide) (PLGA)

hydroxyl (OH), Poly(lactide-coglycolide) carboxylic (PLGA) acid (COOH) modified MWCNTs

CNTs MWCNTs

Cells/models

References

-

MC3T3-E1 osteoblasts

[36]

-

MC3T3-E1 osteoblasts,

[37]

MWCNTs

Poly(L-lactic acid) hydroxyapatite (PLLA)

periodontal ligament cells (PDLCs)

[39]

CNTs

Poly(L-lactic acid) montmorillonite (PLLA) (MMT)

MG-63 cells

[40]

MWCNTs

Poly(L-lactic acid) (PLLA)

hFOB 1.19 cells

[41]

MWCNTs

polyvinyl alcohol (PVA)

biphasic calcium phosphate (BCP)

[42] MC3T3-E1 subclone 14 preosteoblasts derived from newborn mouse calvaria

CNTs

polyvinyl alcohol (PVA)

-

Vero fibroblast-type [43] cells, rat bone marrow-derived mesenchymal stem cells (rBMSCs)

MWCNTs

poly(ether ether calcium ketone) (PEEK) polyphosphate

MC3T3-E1 osteoblast cells

MWCNTs

polymer polypropylene (PP)

Human osteoblast [45] cell line (Saos-2)

hydoxyapatite nanorods (nHAs)

[44]

(Continued)

230  Functionalized CNT for Biomedical Applications Table 10.1  CNTs based composite for bone tissue engineering. (Continued) CNTs

Composite material

Functionalized ligand

SWCNTs

Chitosan

MWCNTs

Cells/models

References

nHAP

human fetal osteoblasts (CRL-11372)

[46]

Chitosan

HAP

MG-63 cells

[47]

MWCNTs

Collagen

HAP

bone marrowderived MSCs (BMSCs)

[48]

Functionalized MWCNTs

Collagen

HAP, chitosan

MG-63 cells

[49]

Functionalized MWCNTs

Alginate

HAP

MG-63 cells

[50]

MWCNTs

bacterial cellulose (BC)

amphiphilic comb-like polymer (APCLP)

critical-sized defect in mouse calvaria

[51]

MWCNTs

Silk fibroin (SF)

nHAP, BMSCs polyamide 66, dexamethasone

[52]

In view of this background, this chapter gives a detailed description of the recent advances on the CNTs based biomaterials for bone tissue engineering as listed in Table 10.1 [16]. Further, mechanisms behind the superior bone tissue repair and regeneration using CNTs based materials are also discussed at the molecular level. Eventually, the need for thorough toxicological studies, pharmacokinetics, and pharmacodynamic investigations is emphasized before the novel materials can enter the real-time therapeutic practice.

10.2 CNT-Based Scaffolds and Implants Biomaterials used to fabricate scaffolds for bone tissue engineering should be rigid to bear external force. Rough surfaces of such materials can promote better cell adhesion and proliferation. Porous structures of

CNTs for Bone Tissue Engineering  231 the materials can help in better penetration of the body fluids and help in neovascularisation. Biodegradable materials are more preferred to avoid the need for surgical removal of the implant after healing the injury. Hence, CNTs with attractive physical and chemical properties are most preferred for the effective reinforcements of the scaffolds in combination with polymers such as collagen, gelatine, alginate, chitosan, and others [17]. Further, CNTs exhibit superior biocompatibility, which in combination with poly(L-lactide) displayed high direct-current conductivity, crystallization, plasticization of the polymer matrix, and growth inhibition of fibroblast cells [18]. Likewise, CNTs can also be encapsulated by poly(lactic acid) (PLA) nanofibers employing electrospinning [19]. Hence, this section elaborates on the CNTs based scaffolds and implants for bone tissue engineering.

10.2.1 Hydroxyapatite Among several calcium phosphate bioceramics which are widely used for orthopedic and dental treatment, hydroxyapatite (HAP) nanoparticle Ca10(PO4)6(OH)2 is used as the main constituent as its predominant naturally in bones and teeth [20]. Nanostructured HAP is biocompatible and used in combination with CNTs to develop scaffolds with high tensile strength and toughness for load-bearing orthopedic implants [21]. A sol-gel method was used by Hooshmand et al. where initially (NH4)2HPO4 solution was added to CNTs sol followed by drop-wise addition of (Ca(NO3)2.4H2O) [22]. Aging for 24 h resulted in the formation of the MWCNT/HAP nanocomposite. It is important to note that the HAP particles were homogenous with a spherical shape that formed clusters, while MWCNTs were dispersed within the clusters without any visible agglomeration. Low temperature for crystallization promoted the formation of more nucleation sites that prevented both dehydroxylation of HAP and oxidation of nanotubes. Further, SDS-adsorbed MWCNTs provided more negative charge on the surface and hence were completely covered with HAP crystal. Cellular attachment and efficient proliferation of the Human bone marrow stromal cells (hBMSC) cells on the MWCNT/HAP nanocomposite indicated biocompatibility of the scaffolds and the bone tissue engineering potential. In a recent study, Jyoti et al. (2021) fabricated a composite by combining both graphene oxide-carbon nanotube (GCNTs) hybrid and HAP [23]. Initially, the MWCNTs were synthesized using the CVD method by reacting toluene and ferrocene. The resulting MWCNTs

232  Functionalized CNT for Biomedical Applications were further acid-functionalized (denoted as FCNTs) by reacting with 70% nitric acid. Graphene oxide (GO) was synthesized by a modified Hummers method, while the GCNTs hybrid was eventually synthesized by ultra-sonicating a mixture containing both GO and FCNTs in ethanol at a ratio of 1:1. The graphene flakes were rippled and crumpled structures of graphene flakes. Morphological features with elemental

(a)

(d) GCNTs

10µm

(O)

(b)

(e) GCNTs

20µm

(P)

(c)

(f)

(Ca)

(C)

Figure 10.2  (a–b) SEM of GCNTs-HAP composites at different magnifications with the elemental mapping of HAP show the presence of (c) Calcium, (d) Oxygen, (e) Phosphorous, and (f) Carbon. Reprinted with permission from Jyoti J, Kiran A, Sandhu M, Kumar A, Singh BP, Kumar N (2021) Improved nanomechanical and in-vitro biocompatibility of graphene oxide-carbon nanotube hydroxyapatite hybrid composites by synergistic effect. Journal of the Mechanical Behavior of Biomedical Materials 117, 104376. Copyright © 2021 Published by Elsevier Ltd [23].

CNTs for Bone Tissue Engineering  233 mapping of the nanocomposite exhibited strong O, P, and Ca signals attributed to the HAP component as evident from Figure 10.2. High nanoindentation hardness and elastic modulus of GCNTs-HAP indicated a highly reinforced nature that is ideal for mimicking bone tissue microenvironment. High cell viability in Madin-Darby Canine Kidney (MDCK) cells after treatment with GCNTs-HAP composites containing 2% of GCNTs nanofiller reflected the high biocompatibility apart from superior induction of cell proliferation. Hence, GCNTs can be a promising composite for promoting bone repair, regeneration, augmentation, and implantation. Khalid and Suman (2017) reported HAP-reinforced MWCNTs with high biocompatibility. Initially, the MWCNTs were treated with 20% sulphuric acid and 20% nitric acid in a 3:1 ratio for one hour [24]. The washed and dried MWCNTs were then dispersed in Sodium Dodecyl Benzene Sulphonate (SDBS) followed by synthesis of HAP in the same solution. The HAP particles were densely oriented on the MWCNTs surface. The nanocomposite was nontoxic to L929 mouse fibroblast cell line. Martinelli et al. (2018) deposited nanohydroxyapatite/superhydrophilic MWCNTs (nHAP/MWCNT) onto 316L stainless steel biomedical alloys by electrophoretic deposition using a voltage of -2V [25]. The outer and inner diameters of the MWCNTs were 60 nm and 50 nm, respectively. Figure 10.3 shows the morphology of the plate-like crystals of the electrodeposited nHAP on the stainless steel surface. The presence of nHAP in the composite was confirmed by the peaks at 961 cm-1, 1030–1050 cm-1, 420 cm-1, 580 cm-1, and 780 cm-1 in Fourier Transform Infrared (FTIR) spectra. Human osteoblast cells (MG-63) cultivated on nHAp/MWCNT showed upregulation of the expression of important genes such as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) that are associated with bone mineralization and maturation. In an interesting study, Rajesh et al. (2016) reported novel tricomponent composite scaffolds composed of oxidized carbon nanotube (fMWCNT)–gellan–hydroxyapatite (HAP) and fMWCNT–amylopectin– HAP with high porosity of 82.82% and 91.76%, respectively [26]. The pore size of fMWCNT–gellan–HAP and fMWCNT–amylopectin–HAP were 49–51  µm and 55–85 µm, respectively, as evident from Figure 10.4. The HAP was uniformly dispersed on the polysaccharide-covered scaffolds. The biomaterial efficiently promoted the MG63 cell adhesion, proliferation, and mineralization.

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Figure 10.3  SEM and optical profilometry collected from deposited nHAp and nHAp/ MWCNT films on stainless steel alloys. (a1) SEM illustrated plate-like crystals of nHAp electrodeposited onto 316L stainless steel alloy; (a2) optical images from profilometry collected at the top of nHAp thin films electrodeposited onto 316L stainless steel; (e3) 3D constructions extracted from the top of 316L stainless steel alloy; (b1) SEM illustrating needle-like crystals electrodeposited onto 316L stainless steel alloy; (b2) optical images from profilometry collected at the top of nHAp/MWCNT thin films electrodeposited onto 316L stainless steel; (c3) 3D constructions collected from the top of 316L stainless steel alloy illustrating nHAp/MWCNT 1% electrodeposited onto 316L stainless steel; (c1) SEM illustrated needle-like crystals electrodeposited onto 316L stainless steel alloy; (c2) optical images from profilometry collected at the top of nHAp/MWCNT thin films electrodeposited onto 316L stainless steel; (c3) 3D constructions extracted from the top of 316L stainless steel alloy illustrating nHAp/MWCNT 3% electrodeposited onto 316L stainless steel. SEM scale bars from Figure 10.3(a1,b1,c1) is 2 µm. Reprinted with permission from Martinelli NM, Ribeiro MJG, Ricci R, Marques MA, Lobo AO, Marciano FR (2018) In vitro osteogenesis stimulation via nano-hydroxyapatite/carbon nanotube thin films on biomedical stainless steel. Materials, 11, 1555 [25]. (Open access).

10.2.2 Polymers Several polymers are incorporated with CNTs for developing scaffolds for bone tissue engineering. In this section, such polyester materials are discussed that are flexible, biodegradable, and biocompatible.

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Figure 10.4  FE-SEM images of (a & b) HAP, and the (c & d) fMWCNT–gellan–HAP and (e & f) fMWCNT–amylopectin–HAP scaffolds. Reprinted with permission from Rajesh R, Ravichandran YD, Reddy MJK, Ryu SH, Shanmugharaj AM (2016) Development of functionalized multi-walled carbon nanotube-based polysaccharide–hydroxyapatite scaffolds for bone tissue engineering. RSC Adv. 6, 82385–82393. Copyright © 2016 The Royal Society of Chemistry [26].

10.2.2.1 Poly(ε-Caprolactone) Huang et al. (2019) fabricated 3D printed scaffolds with the poly(ε-caprolactone) or (PCL) loaded with MWCNTs at different concentrations (0.25, 0.75, and 3 wt%) for bone tissue regeneration [27]. The pore size of the

236  Functionalized CNT for Biomedical Applications scaffolds was in a range from 366 μm and 397 μm. Contact angles in all of the scaffolds were higher than 90° indicating hydrophobic nature that was attributed to the intrinsic hydrophobicity of PCL. The cellular viability of human adipose-derived stem cells (hADSCs) was more than 80% in the PCL-based scaffolds and showed high cell density and cell bridging after 21 days, as shown in Figure 10.5. Mostafavi et al. (2021) engineered composite filaments from a combination of PCL doped with zinc oxide nanoparticles (ZnO NPs) and hydroxyapatite microparticles for in situ bone printing. These scaffolds could adhere to wet bone tissue to prevent dislocation, and over time they showed osteoconductive and supported the osteodifferentiation of mesenchymal stem cells [28]. In another study, Mattioli-Belmonte et al.

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CNTs for Bone Tissue Engineering  237 (2012) microfabricated CNTs–PCL composite exhibited promising bone tissue regenerating effect by promoting proliferation in osteoblast-like cells (MG63) [29]. Interestingly the elastic modulus initially increased with increasing concentration of CNTs in PCL concentration until 12.5 mg/mL, beyond which it started to decrease. The elastic modulus of the nanocomposite varied between 10 and 75 MPa. The composites were more porous and irregular, which facilitated the evenly spreading of the osteoblasts. Similar composite scaffolds also facilitated the attachment and proliferation of the rat bone marrow-derived mesenchymal stem cells (rBMSCs) isolated from young (4–6 weeks old) Sprague Dawley rats. The cells were tightly anchored on the surface of the scaffolds and showed a fibroblast-like morphology, indicating that cells had successfully attached and were effectively spreading [30]. Similarly, amine-functionalized single-walled carbon nanotubes (aSWCNTs)-PCL composite-based electrospun scaffolds promoted the attachment, proliferation, differentiation of rBMSCs. It is important to note that superior mechanical property was obtained when 0.2 wt% aSWNT was incorporated in the scaffold. However, higher rBMSCs proliferation and differentiation was achieved when 0.5 wt% aSWNT was used [31]. One more study conducted by Yang et al. (2019) reported the osteoproductive potential of a three-phased multiwalled scaffold composed of MWCNTs, nHAP, and PCL [32]. The composite scaffold demonstrated smaller porosity and slow degradability in simulated body fluid (SBF). The higher proliferation of human osteosarcoma cell MG63 was observed on the scaffolds that efficiently differentiated into an osteogenic lineage that was confirmed by the high expression of ALP.

10.2.2.2 Polymethyl-Methacrylate Among various bone cements, polymethyl-methacrylate (PMMA) is the most popularly used material that acts as a promising grouting agent in total joint replacement that promotes efficient load transfer between bones and implant. Composites prepared using PMMA, chitosan (CS) powder, and MWCNTs exhibited superior mechanical properties such as increased compressive and bending strengths [33]. The average size was 7.49 µm. The apatite-like deposition was observed on the surface after 28 days. The MG-63 cells could adhere efficiently to the composite surface and expressed high levels of ALP. Pahlevanzadeh et al. (2021) fabricated a novel bioactive polymethyl methacrylate-hardystonite (PMMA-HT)

238  Functionalized CNT for Biomedical Applications (a) PMMA

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Figure 10.6  SEM images of fracture surfaces for (a) PMMA, (b) PMMA/HT, (c) PMMA/ HT/0.25CNT, (d) PMMA/HT/0.5CNT. Reprinted with permission from Pahlevanzadeh F, Bakhsheshi-Rad HR, Kharaziha M, Kasiri-Asgarani M, Omidi M, Razzaghi M, Ismail AF, Sharif S, RamaKrishna S, Berto F (2021) CNT and rGO reinforced PMMA based bone cement for fixation of load-bearing implants: Mechanical property and biological response. J. Mech. Behav. Biomed. Mater. 116, 104320. Copyright © 2021 Elsevier Ltd [34].

porous bone cement (BC) reinforced with 0.25 and 0.5 wt% of CNTs, as evident from Figure 10.6 [34]. The incorporation of 0.5% CNTs enhanced the bending strength of PMMA/HT cements from 61.06 ± 0.68 to 82.07 ± 1.01 MPa. Even the tensile strength increased from 40.02 ± 0.71 MPa to 63.21 ± 1.70 MPa due to the presence of 0.5 wt% CNTs. Superior toughening, apatite formation, and cell interaction in CNTs supplemented composites was noticed when MG63 osteoblasts were grown on the bone cement composite indicating better promotion of implant fixation and load-bearing attributes.

10.2.2.3 Poly(Lactide-Co-Glycolide) Poly(lactide-co-glycolide) (PLGA), a co-polymer of poly(lactic acid) (PLA), and poly(glycolic acid) (PGA) is considered as one of the most popular scaffold matrix polymers due to its biocompatibility, osteoconductivity,

CNTs for Bone Tissue Engineering  239 and suitable mechanical properties that are prerequisites for skeletal tissue repair and regeneration [35]. Porous scaffolds were fabricated by solvent casting/particulate leaching after dispersing the CNTs in PLGA solution. The cubic pore structure with 100 and 200 µm diameters was interconnected. Further, the incorporation of 1% CNTs enhanced the roughness of the composite from 7.17 ± 1.62 to 43.95 ± 10.74 nm. Similarly, the compressive modulus also increased by 3.6-fold with an average compressive modulus of 6.36 ± 0.55 MPa in 1% CNTs supplemented scaffolds. Not only the mechanical strength was increased, but also the surface roughness was enhanced, resulting in better adherence and cell division in MC3T3-E1 osteoblasts. The cell number increased around 2.9-fold per mm2 on 1% CNT/PLGA films. Additionally, the same films enhanced the osteogenic differentiation associated with the highest level of ALP expression. Notably, 1.5-fold increase in the calcium deposit was observed after 28 days when the cells were grown on 1% CNT/PLGA scaffolds. In another study, biodegradable PLGA/MWCNTs scaffolds were synthesized using a thermally induced phase separation (TIPS) technique [36]. The scaffolds exhibited porous morphology with interconnected cavities between 50 and 75 μm in size. These cavities were ideal for harboring cells of a specific type. Around 60% mass degradation was observed for the PLGA/MWCNTs 0.5%. It is important to note that the viability of MC3T3-E1 pre-osteoblast cell line reduced less than 30% in the presence of PLGA/MWCNTs composite with 0.1% of MWCNTs while 0.3 and 0.5% of MWCNTs in scaffolds showed high cytotoxicity. Likewise, Mikael et al. (2014) developed PLGA based 3D porous scaffolds with pristine and modified (with hydroxyl (OH), carboxylic acid (COOH)) MWCNTs [37]. Mechanical properties such as compressive strength and modulus were significantly increased up to 35 MPa and 510.99 MPa, respectively, in the presence of 3% MWCNTs in the composite. The MC3T3-E1 cells exhibited superior viability, proliferation, and mineralization on the composite scaffolds. Inflammatory response after implantation was minimum with OH-modified MWCNTs. In-vivo studies were carried out by implanting the scaffolds subcutaneously in 28 male, retired breeder Sprague-Dawley rats of weight 450–500 g. After 4 weeks, the structural integrity of the implants started to decrease, which was attributed to the degradation of the polymer and invasion by host tissues which was more prominent by 8-12 weeks. A mild response and development of a fibrous capsule composed of a few lymphocytes, giant cells, polymorphonuclear (PMN) lymphocytes, and mainly macrophages were observed for the 3% OH-MWCNT supplemented scaffold.

240  Functionalized CNT for Biomedical Applications

10.2.2.4 Poly-L-Lactic Acid Poly(L-lactic acid) (PLLA) is another polymer with superior biocompatibility that is used for designing scaffolds for tissue engineering [38]. In one such study, Mei et al. (2007) fabricated a composite membrane for guided tissue regeneration (GTR) by electrospinning a suspension consisting of PLLA, MWCNTs, and HAP [39]. The composite enhanced the adhesion and proliferation of periodontal ligament cells (PDLCs) by 30% and inhibited the adhesion and proliferation of gingival epithelial cells by 30%. On implantation of the composite membrane seeded PDLCs into the leg muscle pouches of immunodeficient mice. Interestingly, the composite promoted the formation of round to irregular bone with a higher calcium deposit. Further, the porous structure of the scaffolds facilitated the growth of the blood vessels that played a critical role in bone tissue regeneration. Shuai et al. (2021) developed a self-assembled hybrid nanomaterial from acidified CNTs, aminopropyltriethoxysilane (KH550) [40]. The ammonium salt grafted CNTs successfully intercalated into the interlayers of large, flat, and layered montmorillonite (MMT). This was mainly attributed to the cation exchange reaction between the alkylammonium cations and the sodium cations of MMT. The MMT-CNT hybrid was then incorporated into PLLA that significantly increased the tensile strength and modulus by 113.04% and 111.46%, respectively. The composite scaffolds with MMT-CNT contents of 3%, 6%, 9%, and 12% were termed as PMC3, PMC6, PMC9, and PMC12, respectively. Likewise, the compressive strength and modulus were enhanced by 58.20% and 63.27%, respectively. Figure 10.7 shows that compared to PLLA, the MMT-CNT containing scaffolds are darker. The uniformly distributed pores in the scaffolds are advantageous as they can facilitate better cellular growth by allowing efficient transportation of nutrients, oxygen, and other essential molecules. Moreover, incorporation of the MMT-CNT in a varied amount ranging from 0% to 12% increased the water absorption rate of the scaffolds from 130.02% to 169.23%. Higher cell adhesion, growth, and proliferation of MG-63 cells were seen on the PLLA based scaffolds. Likewise, Vozzi et al., 2013 reported microfabricated three-dimensional (3D) scaffolds composed of PLLA and MWCNTs that were biocompatible to the hFOB 1.19 cells. The cells exhibited better anchoring on the composite scaffold surface. However, the osteoblasts showed more spheroid morphology [41].

CNTs for Bone Tissue Engineering  241 PMC3

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Figure 10.7  Digital and SEM images in different magnifications of the PLLA and PMC scaffolds. Reprinted with permission from Shuai C, Peng B, Liu M, Peng S, Feng P (2021) A self-assembled montmorillonite-carbon nanotube hybrid nano-reinforcement for poly-L-lactic acid bone scaffold. Mater. Today Adv. 11, 100158. Copyright © 2021 The Author(s). Published by Elsevier Ltd [40]. .

10.2.2.5 Polyvinyl Alcohol As an integral part, the tissue engineering scaffolds have a supporting material that should be non-toxic, biocompatible, and stable. One such mechanically suitable supporting material with biotribological properties is polyvinyl alcohol (PVA) which is extensively studied for developing osteochondral scaffolds. Lan et al. (2019) developed an MWCNTs reinforced polyvinyl alcohol/biphasic calcium phosphate (PVA/BCP) scaffold employing a freeze-thawing and freeze-drying method [42]. The composite scaffold showed an enhanced compressive strength of 81 ± 6 kPa as compared to only PVA/BCP hydrogels (48 ± 2 kPa). The porous scaffolds exhibited high interconnectivity equivalent to 80 ± 0.6%. The viability of MC3T3-E1 subclone 14 preosteoblasts derived from newborn mouse calvaria was high on the composite PVA/BCP/CNT porous scaffolds. Further, the scaffold surface promoted high proliferation and spreading of the cells confirming the tissue regenerating potential.

242  Functionalized CNT for Biomedical Applications Yet, in another study CNTs and carbon nanofibres (CNF) were produced by hot-filament chemical vapor deposition (HFCVD) for reinforcement of PVA-based hydrogel [43]. High biocompatibility was observed when the Vero fibroblast-type cells’ were grown on the scaffolds. Interestingly, the composite induced significant osteogenic differentiation in the MSCs derived from rat bone marrow, as indicated by high alkaline phosphatase (ALP) activity and mineralization. Hence, it was speculated that the CNTs reinforced PVA hydrogels can be effectively used to treat osteochondral defects.

10.2.2.6 Others Certain other biocompatible polymers are considered suitable for developing scaffolds for bone tissue engineering. Poly(ether ether ketone) (PEEK) is one of the most preferred polymers that can replace conventional surgical metal implants owing to its mechanical properties and stiffness identical to native bone tissues. In one such study, Cao et al. (2018) fabricated a bioactive composite made from MWCNTs, calcium polyphosphate, and PEEK [44]. Superior interfacial adhesion resulted in a uniform dispersion of the MWCNTs in the PEEK matrix. Interestingly, the MC3T3-E1 osteoblast cells exhibited high viability and better adhesion on the composite surface. Moreover, the composite promoted osteogenic differentiation of MC3T3-E1 cells that was confirmed by significantly high ALP activity. Likewise, Liao et al. (2013) reported another polymer polypropylene (PP) based biocomposites reinforced by MWCNTs and hydroxyapatite nanorods (nHAs) [45]. The MWCNTs were well dispersed in the polymeric matrix of the nanocomposites, as marked by the black arrows in Figure 10.8. Both the nHA and MWCNTs fillers might compete with each other for the nucleation of PP macromolecules. The MWCNTs promoted nucleation of PP in the composites containing 8% and 15% nHA, particularly the former with low nHA content. The incorporation of MWCNTs further enhanced the stiffness, tensile strength, and impact toughness of the PP/nHA nanocomposites. The human osteoblast cell line (Saos-2) was firmly attached to the biocomposite surface, and rapid proliferation led to covering the entire surface after 3 days. Interestingly, osteoblastic proliferation was found to be a function of the culturing time.

10.2.3 Biopolymers Polymers such as chitosan, collagen, fibrin, hyaluronic acid, and alginate of biological origin called biopolymers are very significant due to their high

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Figure 10.8  SEM morphologies for (a) PP/0.1%MWCNT–8%nHA, (b) PP/0.1%MWCNT– 20%nHA, (c) PP/0.3%MWCNT–8%nHA, (d, e) PP/0.3%MWCNT–20% nHA nanocomposites. MWCNTs in these micrographs are denoted with black arrows. Dash square shows nHA fillers together with their EDX spectrum. (f) TEM micrograph of hydroxyapatite nanorods. Reprinted with permission from Liao CZ, Li K, Wong HM, Tong WY, Yeung KW, Tjong SC (2013) Novel polypropylene biocomposites reinforced with carbon nanotubes and hydroxyapatite nanorods for bone replacements. Mater. Sci. Eng. C Mater. Biol. Appl. 33, 1380–1388. Copyright © 2012 Elsevier B.V [45].

244  Functionalized CNT for Biomedical Applications water content, good permeability for oxygen, nutrients, and other soluble signals. Several hydrogel scaffolds are designed using biopolymers as they can provide suitable mechanical properties like fibrous extracellular matrix identical to the native bone tissues.

10.2.3.1 Chitosan Chitin is a biopolymer abundantly available in the exoskeletons of crustaceans and insects. Deacetylation of this chitin results in the formation of chitosan, which has promising therapeutic potential and is used in wound dressings and various tissue engineering applications. Being biodegradable porous chitosan sponges are used for fabrication of scaffolds for promoting the proliferation of osteoblasts. Im et al. (2012) reported 3D porous chitosan scaffolds with different concentrations of nHAP and SWCNTs employing a lyophilization procedure [46]. Initially, two types of SWCNTs were synthesized in an arc discharge with a magnetic field (B-SWCNT) and without a magnetic field (N-SWCNT) for reinforcement purposes to facilitate bone regeneration. Interestingly, the swelling ratios of all composite hydrogels exceeded 600%. The composite scaffolds supported the efficient attachment of the human fetal osteoblasts (CRL-11372), which exhibited long filopodia cell growth spreading onto the surfaces. In another similar study, Venkatesan et al. (2011) employed a freezedrying method to synthesize composite using chitosan and natural hydroxyapatite derived from Thunnus obesus bone (chitosan/HAP) and chitosan grafted with functionalized MWCNTs with HAP (f-MWCNT-gchitosan/HAP) [47]. A notable reduction in the water uptake, retention ability, and degradation of composite scaffolds was observed. However, the thermal stability of the composite increased on incorporation of HAP and f-MWCNT. Equal porous structures with good interconnectivity were observed in Figure 10.9. Both HAP and f-MWCNT were homogeneously dispersed in the chitosan matrix with interconnected porosity of 70–200 µm(chitosan/HAP) and 46–200 µm(f-MWCNT-g-chitosan/HAP). Proliferation in MG-63 cells was doubled when cultured on the composite surface.

10.2.3.2 Collagen Another naturally occurring biopolymer is collagen (Col) which shows low antigenicity, high biocompatibility, and provides the most favorable condition for cell attachment, proliferation, and differentiation for neurite, osteoblasts and fibroblasts. Jing et al. (2017) fabricated an innovative

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Figure 10.9  SEM images of (a) chitosan scaffold (b) chitosan/HAP scaffold (c) f-MWCNT-g-chitosan/HAP scaffold, and (d) dispersion of HAP particles in chitosan matrix. Reprinted with permission from Venkatesan J, Qian Z-J, Ryu B, Kumar NA, Kim SK (2011) Preparation and characterization of carbon nanotube-grafted-chitosan – Natural hydroxyapatite composite for bone tissue engineering. Carbohydr Polym 83, 569–577. Copyright © 2010 Elsevier Ltd [47].

porous scaffold by reinforcing MWCNTs into a Col-HAP composite [48]. This enhanced the stiffness by 10-fold. Further, this scaffold promoted the proliferation and spreading of the bone marrow-derived MSCs (BMSCs) that was confirmed by high levels of expression of bone sialoprotein (BSP) and osteocalcin (OCN). Dense connective tissues covered the defect site,

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Figure 10.10  SEM micrographs of Col, CS, Col/f-MWCNT, and Col/f-MWCNT/CS scaffolds (a) before biomineralization at magnification ×50 and (b) after biomineralization at magnification ×1,000. Reprinted with permission from Türk S, Altınsoy I, GÇ, Ipek M, Özacar M, Bindal C (2018) 3D porous collagen/functionalized multiwalled carbon nanotube/chitosan/hydroxyapatite composite scaffolds for bone tissue engineering. Mater. Sci. Eng. C 92, 757–768. Copyright © 2018 Elsevier B.V [49].

CNTs for Bone Tissue Engineering  247 followed by new bone formation that was triggered by the composite scaffold resulting in effective repair of the rat calvarial defects (8 mm diameter). In another study, Türk et al. (2018) fabricated a novel collagen/functionalized MWCNTs/chitosan/hydroxyapatite composite scaffold represented as Col/f-MWCNT/CS/HA [49]. The composite was developed by freezing at up to −40°C at a 0.9°C/min rate followed by lyophilization (48 h, 0°C, and 200 mtorr). The compressive stress of the scaffold was in a range from 523 to 1112 kPa while swelling varied between 513.9 ± 27 and 481.05 ± 25%. The scaffolds were porous, which is advantageous for transporting the metabolites and nutrients for promoting cellular growth. The porosity ranged from 98 ± 0.15 to 95.7 ± 0.1%. A superior pore volume of 0.026 cc/g with interconnected porous microstructure was noticed, as evident from Figure 10.10. The pore size was in a range from 20 to 350 μm. The contact angle was between 87.8 and 76.7°. Further, the scaffolds showed a large surface area equivalent to 11.746 m2/g. High viability of the MG-63 cells was noticed when grown on the composite scaffolds.

10.2.3.3 Others Some unusual polymers are also explored for tissue engineering purposes. Seaweed-derived polysaccharide, alginate is mainly composed of 1,4-linked β-D-mannuronic acid and α-L-guluronic acid residues. This biomaterial can get cross-linked in the presence of divalent cations such as Ca2+, resulting in gel formation. Alginate is considered as a significant biopolymer for drug delivery, wound dressings, and tissue engineering. In one such study, Rajesh and Ravichandran (2015) designed a tricomponent scaffold comprised of alginate, oxidized MWCNTs, and HAP using a freeze-drying method [50]. The HAP used in this scaffold was naturally derived from the chicken bones by thermal calcination at 800°C. The resulting fMWCNT– alginate–hydroxyapatite scaffold exhibited interconnected porosity with a pore size of 130–170 μm while the total porosity was 93.85%. The scaffold was biocompatible to MG-63 cells and did not show any toxicity. Instead, the composite scaffold promoted better cell proliferation, cell differentiation, and cell attachment in MG-63 cells. In another study, Park et al. (2015) fabricated a 3D scaffold using bacterial cellulose (BC) incorporated with MWCNTs [51]. Initially, the bacteria Gluconacetobacter xylinus, was pre-cultured on a medium composed of 2.5% (w/w) mannitol, 0.5% (w/w) yeast extract, and 0.3% (w/w) bacto-peptone and eventually introduced in MWCNTs-dispersed culture medium at a ratio of 1:10. The reaction mixture was then incubated for 14

248  Functionalized CNT for Biomedical Applications days at 28°C. The composite membrane composed of MWCNTs and BC was then harvested and purified by treatment with boiling 1 wt% sodium hydroxide for 2 h. Further, an amphiphilic comb-like polymer (APCLP) was adsorbed on the scaffold resulting in the formation of the MWCNTBC-Syn with a core-shell structure, where MWCNTs were packed by BC nanofibril entanglements. The BC nanofibrils were 4.3 nm thick while around 3.9% of the MWCNTs surface was uncovered. Efficient regeneration of the bone tissue was noted when the scaffolds were implanted in the critical-sized defect in mouse calvaria. Silk fibroin (SF) is another natural polymer extracted from silk fibrous protein. Its superior mechanical properties, tensile strength, and flexibility make it promising biomaterial tissue engineering. Moreover, its biodegradable nature, higher permeability, and slow-releasing potential make it ideal for biomedical applications. It is also advantageous for bone tissue regeneration as it can induce bone mineralization due to the predominance of carboxylic acid groups in its side chains. Yao et al. (2016) synthesized a nano-hydroxyapatite/polyamide 66 (nHAP/PA66) porous scaffold employing a phase inversion method and surface modified with which MWCNTs and SF using freeze-drying and cross-linking [52]. The resulting MWCNT/SF-nHA/PA66 scaffolds had an average diameter of ~500 μm while the porosity and compressive strength were 62.07% and 5.4 MPa, respectively. The water-absorbing rate of the scaffold was 49.11% after CNT/SF modification. In order to promote osteogenic differentiation in BMSCs, dexamethasone (DEX) was functionalized on the MWCNTs. The cumulative amount of DEX release was 97% in 0.5% SDS and 66% in PBS within 24h. Interestingly, large holes within the scaffolds were colonized by some of the cells, and DEX-loaded MWCNT/SF-nHA/PA66 scaffold induced more ALP secretion.

10.3 Intellectual Property Rights and Commercialization Aspects Innovation in the area of functionalized CNTs has led to efficient commercially viable product development with potential marketability, as listed in Tables 10.2 and 10.3. In one such attempt, marine bioprocess waste, chitosan/hydroxyapatite (CNT-gCTS/HAp) complex scaffold was conjugated with CNTs to develop a bioactive material for bone tissue transplantation. The material showed promising thermal stability, interconnected pores, controlled biodegradability, and non-cytotoxicity [KR101273536B1] [53].

CNTs for Bone Tissue Engineering  249 Table 10.2  Patents on CNTs based biomaterials for bone tissue engineering. Publication date

Reference

Carbon nanotubegrafted-chitosanhydroxyapatite composite for bone tissue engineering and a method of preparing the same

2013-06-14

[53]

KR101309416B1

Carbon nanotubechitosan-silica nanohybrid for bone regeneration and method of preparation thereof

2013-09-23

[54]

KR101663350B1

CNT-gelatinhydroxyapatite nanohybrids with fibrous multilayer core-shell structure for mimicking natural bone

2016-10-06

[55]

CN106178100A

Carbon Nanotubes/ Chitosan complex microsphere surface forms the preparation method of orientation nano-apatite

2021-06-08

[56]

CN107537063B

A kind of complex stephanoporate bracket of carbon nanotubes and preparation method thereof

2019-10-11

[57]

Patent number

Title

KR101273536B1

(Continued)

250  Functionalized CNT for Biomedical Applications Table 10.2  Patents on CNTs based biomaterials for bone tissue engineering. (Continued) Publication date

Reference

Carbon nanotubegrafted-chitosanhydroxyapatite composite for bone tissue engineering and a method of preparing the same

2012-05-23

[58]

Carbon nanotube composite scaffolds for bone tissue engineering

2013-12-24

[59]

Patent number

Title

KR20120052116A

US8614189B2

Table 10.3  Companies manufacturing scaffolds. Name of company

Website

Ref.

LAYHER PTE LTD, Singapore

http://www.layher.sg/?gclid=Cj0KCQiA jc2QBhDgARIsAMc3SqSIKjWV gefEavamSBUTphH0i70QNju-tkz2JR GGChwKpvWvIcKI8YEaAu7 OEALw_wcB

[60]

NORD, China

https://www.nordscaffolding.com/about-us/

[61]

YANCHENG LIANGGONG FORMWORK CO., LTD., China

https://lianggongform.en.made-inchina.com/?keyword=scaffolding %20supplier&gclid=Cj0KCQiAjc2 QBhDgARIsAMc3SqQxcT35smk5w N0qcWMZnUlGyi5L7eLGkLv71JpckJ zqotI98xjoGvIaAm6OEALw_wcB

[62]

Dura, China

https://www.durascf.com/about/

[63]

CNTs for Bone Tissue Engineering  251 In another patent, a CNTs-chitosan-silica hybrid nanocomposite exhibited superior osteogenic potential, mechanical strength, and biocompatibility [KR101309416B1] [54]. CNTs-gelatin-hydroxyapatite based composite biomaterial was patented that showed a multilayer core-cell fiber bone mimetic structure [KR101663350B1] [55]. CNTs/chitosan complex microsphere were fabricated for an efficient orientation of nanoapatite that was speculated to be an efficient bone renovating biomaterial [CN106178100A] [56]. Chitosan, gelatin, hydroxyapatite, along with CNTs served as an excellent biomedical material for bone tissue engineering, clinical transplantation, and reparation [CN107537063B] [57]. Some scaffold manufacturing companies have started commercial synthesis of scaffolds for biomedical applications, as listed in Table 10.3.

10.4 Conclusion and Future Perspectives Developing scaffolds for bone tissue engineering is challenging as the biomaterial should be able to provide adequate mechanical strength to the newly formed bone tissues. Hence, various nanomaterials are explored with superior reinforcement attributes. CNTs are considered as a viable solution to generate superior biomaterials that can be used for hard tissue engineering. However, the long-term effect of the CNTs on the target tissues should be thoroughly investigated. Similarly, it is necessary to exclusively study the biodistribution of the CNTs before they can be used in real-time therapy for bone tissue regeneration. Further, the physical properties such as length, thickness, and rigidity of the CNTs largely determine the toxic effects, and hence rational modification may help in developing nanostructures with minimum or no health hazards. The large surface area of the CNTs can be rationally functionalized with the drugs and biomolecules. This may help trigger the cellular response in the site of injury, hence promoting more cell division, differentiation and mineralization. At the same time, functionalization with the contrast agents may help in bioimaging for effective monitoring of the healing process. Antimicrobial drugs or nanoparticles can be impregnated in the scaffolds to prevent the post-surgical risks of bacterial infections [64]. The drug release profiles should be carefully studied by selecting an array of chemical and biological polymers used for fabricating the scaffolds. However, thorough pharmacokinetics and pharmacodynamics studies should be carried out to ensure the safety of the novel CNTs based biomaterials that have a huge potential for revolutionizing regenerative medicine.

252  Functionalized CNT for Biomedical Applications

References 1. Trzeciak, T. et al., Cells and nanomaterial-based tissue engineering techniques in the treatment of bone and cartilage injuries. J. Nanosci. Nanotechnol., 16, 9, 8948–8952, 2016. 2. Mehranfar, S. et al., The use of stromal vascular fraction (SVF), platelet-rich plasma (PRP) and stem cells in the treatment of osteoarthritis: An overview of clinical trials. Artif. Cells Nanomed. Biotechnol., 47, 1, 882–890, 2019. 3. Spin-Neto, R. et al., Clinical similarities and histological diversity comparing fresh frozen onlay bone blocks allografts and autografts in human maxillary reconstruction. Clin. Implant Dentist. Relat. Res., 15, 4, 490–497, 2013. 4. Amorosa, L. et al., Physiologic load-bearing characteristics of autografts, allografts, and polymer-based scaffolds in a critical sized segmental defect of long bone: An experimental study. Int. J. Nanomedicine, 8, 1637, 2013. 5. Roberts, T.T. and Rosenbaum, A.J., Bone grafts, bone substitutes and orthobiologics: The bridge between basic science and clinical advancements in fracture healing Organogenesis, 8, 4, 114–124, 2012. 6. Fernandez-Bances, I. et al., Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft. Cytotherapy, 15, 5, 571–577, 2013. 7. Pei, B. et al., Applications of carbon nanotubes in bone tissue regeneration and engineering: Superiority, concerns, current advancements, and prospects. Nanomaterials, 9, 10, 1501, 2019. 8. Huang, B., Carbon nanotubes and their polymeric composites: The applications in tissue engineering. Biomanuf. Rev., 5, 1, 1–26, 2020. 9. Mostafavi, E., Soltantabar, P., Webster, T.J., Nanotechnology and picotechnology: A new arena for translational medicine, in: Biomaterials in Translational Medicine, pp. 191–212, Elsevier, Netherlands, 2019. https://doi.org/10.1016/ C2016-0-04209-2 10. Ghosh, S. and Webster, T.J., Metallic nanoscaffolds as osteogenic promoters: Advances, challenges and scope. Metals, 11, 9, 1356, 2021. 11. Mostafavi, E. et al., Electroconductive nanobiomaterials for tissue engineering and regenerative medicine. Bioelectricity, 2, 2, 120–149, 2020. 12. Ghosh, S. and Webster, T.J., Mesoporous silica based nanostructures for bone tissue regeneration. Front. Mater., 8, 213, 2021. 13. Zare, H. et al., Carbon nanotubes: Smart drug/gene delivery carriers. Int. J. Nanomedicine, 16, 1681–1706, 2021. 14. Haniu, H. et al., Basic potential of carbon nanotubes in tissue engineering applications. J. Nanomater., 2012, 4, 4, 2012. https://doi.org/ 10.1155/2012/343747 15. Medina-Cruz, D. et al., Green nanotechnology-based drug delivery systems for osteogenic disorders. Expert Opin. Drug Deliv., 17, 3, 341–356, 2020. 16. Peng, Z. et al., Bone tissue engineering via carbon-based nanomaterials. Adv. Healthc. Mater., 9, 5, 1901495, 2020.

CNTs for Bone Tissue Engineering  253 17. Abarrategi, A. et al., Multiwall carbon nanotube scaffolds for tissue engineering purposes. Biomaterials, 29, 1, 94–102, 2008. 18. Zhang, D. et al., Poly (L-lactide) (PLLA)/multiwalled carbon nanotube (MWCNT) composite: Characterization and biocompatibility evaluation. J. Phys. Chem. B, 110, 26, 12910–12915, 2006. 19. McCullen, S.D. et al., Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells. Int. J. Nanomedicine, 2, 2, 253, 2007. 20. Truong, L.B. et al., Advances in 3D-printed surface-modified Ca-Si bioceramic structures and their potential for bone tumor therapy. Mater. (Basel), 14, 14, 3844, 2021. https://doi.org/10.3390/ma14143844 21. Perkins, B.L. and Naderi, N., Suppl-3, M7: Carbon nanostructures in bone tissue engineering. Open Orthop. J., 10, 877, 2016. 22. Hooshmand, T. et al., Development of sol-gel-derived multi-wall carbon nanotube/hydroxyapatite nanocomposite powders for bone substitution. J. Compos. Mater., 48, 4, 483–489, 2014. 23. Jyoti, J. et al., Improved nanomechanical and in-vitro biocompatibility of graphene oxide-carbon nanotube hydroxyapatite hybrid composites by synergistic effect. J. Mech. Behav. Biomed. Mater., 117, 104376, 2021. 24. Khalid, P. and Suman, V., Carbon nanotube-hydroxyapatite composite for bone tissue engineering and their interaction with mouse fibroblast L929 in vitro. J. Bionanoscience, 11, 3, 233–240, 2017. 25. Martinelli, N.M. et al., In vitro osteogenesis stimulation via nanohydroxyapatite/carbon nanotube thin films on biomedical stainless steel. Materials, 11, 9, 1555, 2018. 26. Rajesh, R. et al., Development of functionalized multi-walled carbon nanotube-based polysaccharide-hydroxyapatite scaffolds for bone tissue engineering. RSC Adv., 6, 85, 82385–82393, 2016. 27. Huang, B. et al., Fabrication and characterisation of 3D printed MWCNT composite porous scaffolds for bone regeneration. Mater. Sci. Eng. C, 98, 266–278, 2019. 28. Mostafavi, A. et al., In situ printing of scaffolds for reconstruction of bone defects. Acta Biomater., 127, 313–326, 2021. 29. Mattioli-Belmonte, M. et al., Tuning polycaprolactone-carbon nanotube composites for bone tissue engineering scaffolds. Mater. Sci. Eng. C, 32, 2, 152–159, 2012. 30. Pan, L. et al., Multiwall carbon nanotubes/polycaprolactone composites for bone tissue engineering application. Colloids Surf. B Biointerfaces, 93, 226– 234, 2012. 31. Tohidlou, H. et al., Amine-functionalized single-walled carbon nanotube/ polycaprolactone electrospun scaffold for bone tissue engineering: in vitro study. Fiber. Polym., 20, 9, 1869–1882, 2019. 32. Yang, H., Li, J., Liao, Q., Guo, H., Chen, H., Zhu, Y., Cai, M., Lv., H., In vitro evaluation of a novel multiwalled carbon nanotube/nanohydroxyapatite/

254  Functionalized CNT for Biomedical Applications polycaprolactone composite for bone tissue engineering. J. Mater. Res., 34, 4, 532–544, 2019. 33. Eil Bakhtiari, S.S., Karbasi, S., Tabrizi, S.A.H., Ebrahimi-Kahrizsangi, R., Salehi, H., Evaluation of the effects of chitosan/multiwalled carbon nanotubes composite on physical, mechanical and biological properties of polymethyl methacrylate-based bone cements. Mater. Technol., 35, 5, 267–280, 2020. 34. Pahlevanzadeh, F., Bakhsheshi-Rad, H.R., Kharaziha, M., Kasiri-Asgarani, M., Omidi, M., Razzaghi, M., Ismail, A.F., Sharif, S., Krishna, S.R., Berto, F., CNT and rGO reinforced PMMA based bone cement for fixation of load bearing implants: Mechanical property and biological response. J. Mech. Behav. Biomed. Mater., 116, 104320, 2021. https://doi.org/10.1016/j. jmbbm.2021.104320 35. Cheng, Q., Rutledge, K., Jabbarzadeh, E., Carbon nanotube-poly (lactide-co-glycolide) composite scaffolds for bone tissue engineering applications. Ann. Biomed. Eng., 41, 5, 904–916, 2013. 36. Diaz, E. et al., Hydrolytic degradation and cytotoxicity of poly (lactic-co-glycolic acid)/multiwalled carbon nanotubes for bone regeneration. J. Appl. Polym. Sci., 137, 10, 48439, 2020. 37. Mikael, P.E., Amini, A.R., Basu, J., Arellano-Jimenez, M.J., Laurencin, C.T., Sanders, M.M., Carter, C.B., Nukavarapu, S.P., Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: Fabrication, in vitro and in vivo evaluation. Biomed. Mater., 9, 3, p.035001, 2014. https://doi. org/10.1088/1748-6041/9/3/035001 38. Farahani, A., Zarei-Hanzaki, A., Abedi, H.R., Tayebi, L., Mostafavi, E., Polylactic acid piezo-biopolymers: Chemistry, structural evolution, fabrication methods, and tissue engineering applications. J. Funct. Biomater., 12, 4, p71, 2021. https://doi.org/10.1088/1748-6041/9/3/035001 39. Mei, F. et al., Improved biological characteristics of poly (L-lactic acid) electrospun membrane by incorporation of multiwalled carbon nanotubes/ hydroxyapatite nanoparticles. Biomacromolecules, 8, 12, 3729–3735, 2007. 40. Shuai, C. et al., A self-assembled montmorillonite-carbon nanotube hybrid nanoreinforcement for poly-L-lactic acid bone scaffold. Mater. Today Adv., 11, 100158, 2021. 41. Vozzi, G., Corallo, C., Daraio, C., Pressure-activated microsyringe composite scaffold of poly (L-lactic acid) and carbon nanotubes for bone tissue engineering. J. Appl. Polym. Sci., 129, 2, 528–536, 2013. 42. Lan, W. et al., Carbon nanotube reinforced polyvinyl alcohol/biphasic calcium phosphate scaffold for bone tissue engineering. RSC Adv., 9, 67, 38998– 39010, 2019. 43. Rodrigues, A.A. et al., Polyvinyl alcohol associated with carbon nanotube scaffolds for osteogenic differentiation of rat bone mesenchymal stem cells. Carbon, 50, 2, 450–459, 2012.

CNTs for Bone Tissue Engineering  255 44. Cao, J. et al., Bioactive poly (etheretherketone) composite containing calcium polyphosphate and multi-walled carbon nanotubes for bone repair: Mechanical property and in vitro biocompatibility. J. Bioact. Compat. Polym., 33, 5, 543–557, 2018. 45. Liao, C.Z. et al., Novel polypropylene biocomposites reinforced with carbon nanotubes and hydroxyapatite nanorods for bone replacements. Mater. Sci. Eng. C, 33, 3, 1380–1388, 2013. 46. Im, O. et al., Biomimetic three-dimensional nanocrystalline hydroxyapatite and magnetically synthesized single-walled carbon nanotube chitosan nanocomposite for bone regeneration. Int. J. Nanomedicine, 7, 2087, 2012. 47. Venkatesan, J., Qian, Z.-J., Ryu, B.M., Kumar, N.A., Kim, S.-K., Preparation and characterization of carbon nanotube-grafted-chitosanâ-natural hydroxyapatite composite for bone tissue engineering. Carbohydr. Polym., 83, 2, 569– 577, 2011. 48. Jing, Z., Wu, Y., Su, W., Tian, M., Jiang, W., Cao, L., Zhao, L., Zhao, Z., Carbon nanotube reinforced collagen/hydroxyapatite scaffolds improve bone tissue formation in vitro and in vivo. Ann. Biomed. Eng., 45, 9, 2075–2087, 2017. 49. Türk, S. et al., 3D porous collagen/functionalized multiwalled carbon nanotube/chitosan/hydroxyapatite composite scaffolds for bone tissue engineering. Mater. Sci. Eng. C, 92, 757–768, 2018. 50. Rajesh, R. and Ravichandran, Y.D., Development of a new carbon nanotubealginate-hydroxyapatite tricomponent composite scaffold for application in bone tissue engineering. Int. J. Nanomedicine, 10, Suppl 1, 7, 2015. 51. Park, S. et al., In situ hybridization of carbon nanotubes with bacterial cellulose for three-dimensional hybrid bioscaffolds. Biomaterials, 58, 93–102, 2015. 52. Yao, M.-Z. et al., Fabrication and characterization of drug-loaded nanohydroxyapatite/polyamide 66 scaffolds modified with carbon nanotubes and silk fibroin. Int. J. Nanomedicine, 11, 6181, 2016. 53. 중-지, 김.벤., Carbon nanotube-grafted-chitosan- hydroxyapatite composite for bone tissue engineering and a method of preparing the same. South Korea Patent KR101273536B1, 2013. 54. 김해원김정주신원상, Carbonnanotube-chitosan-silica nanohybrid for bone regeneration and method of preparation thereof. South Korea Patent KR101309416B1, 2013. 55. 신원상김해원, CNT-gelatin-hydroxyapatite nanohybrids with fibrous multilayer core-shell structure for mimicking natural bone. South Korea Patent KR101663350B1, 2016. 56. 黄棣杜晶晶胡银春牛璐璐魏延连小洁陈维毅, Carbon nanotubes/chitosan complex microsphere surface forms the preparation method of orientation nano-apatite. China Patent CN106178100A, 2021. 57. 宋克东蒋思宇卢延国李文芳李丽颖刘天庆, A kind of complex stephanoporate bracket of carbon nanotubes and preparation method thereof. China Patent CN107537063B, 2019.

256  Functionalized CNT for Biomedical Applications 58. K. Se-Kwon, R. Bomi J. Benkatasan, C. Zhong-Ji, Carbon nanotube-graftedchitosan- hydroxyapatite composite for bone tissue engineering and a method of preparing the same. South Korea Patent KR20120052116A, 2013. 59. S.G. Kumbar, C.T. Laurencin, S.P. Nukavarapu, Carbon nanotube composite scaffolds for bone tissue engineering. US Patent US8614189B2, 2013. 60. LAYHER PTE LTD, Singapore. Website: http://www.layher.sg/?gclid=Cj0KCQiAjc2QBhDgARIsAMc3SqSIKjWVgefEavamSBUTphH0i70QNju-tkz2JRGGChwKpvWvIcKI8YEaAu7OEALw_wcB. Accessed on 24th February, 2022. 61. NORD, China. Website: https://www.nordscaffolding.com/about-us/ Accessed on 24th February, 2022. 62. YANCHENG LIANGGONG FORMWORK CO., LTD., China Website: https://lianggongform.en.made-in-china.com/?keyword=scaffolding%20 supplier&gclid=Cj0KCQiAjc2QBhDgARIsAMc3SqQxcT35smk5wN0qcWMZnUlGyi5L7eLGkLv71JpckJzqotI98xjoGvIaAm6OEALw_wcB Accessed on 24th February, 2022. 63. Dura, China. Website: https://www.durascf.com/about/ Accessed on 24th February, 2022. 64. Ibrahim, D.M., Sani, E.S., Soliman, A.M., Zandi, N., Mostafavi, E., Youssef, A.M., Allam, N.K., Annabi, N., Bioactive and elastic nanocomposites with antimicrobial properties for bone tissue regeneration. ACS Appl. Bio Mater., 3, 5, 3313–3325, 2020.

11 Application of Functionalized Carbon Nanotubes in Biomimetic/ Bioinspired Systems Mohammad Mobin1*, Ruby Aslam1, Saman Zehra1, Jeenat Aslam2 and Shahidul Islam bhat1 Corrosion Research Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh, India 2 Department of Chemistry, College of Science, Taibah University, Yanbu, Al-Madina, Saudi Arabia

1

Abstract

Over the past billion years, biological systems have developed various ways to achieve optimal bulk and structure unification. The discovery of biological properties has led to an increased interest in studying these materials through modern characterization techniques. Biomimetics is a field of research that allows scientists to mimic the biological properties of organisms for the development of new materials and processes. The complexity of biological materials and their surfaces can be distinguished from one another through their morphological and chemical properties. In nature, hierarchical structures with dimensions of features ranging from the macroscale to the nanoscale are commonly formed to provide properties of interest. Scientists and engineers are now working on developing new engineered materials that can mimic the properties of biological systems. For the past two decades, the development of carbon nanotubes (CNTs)-reinforced composites has been increasing at a fast pace. This paper presents a detailed study on the current breakthroughs in the manufacturing of bio-inspired hybrid materials. Various bio-inspired functionalities of CNT-based composites, including bioadhesives, injectability, antibacterial properties, and degradability applicable to advanced drug delivery systems and medical devices will be discussed. The paper also reviews the future potential of these materials.

*Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (257–280) © 2023 Scrivener Publishing LLC

257

258  Functionalized CNT for Biomedical Applications Keywords:  Bioactive scaffolds, composite materials, CNTs, bio-mimicking

11.1 Introduction The structure and function of biological materials have been continuously refined over millions of years. Through the study of live organisms, researchers can then apply these principles and technologies to manmade materials to emulate biological functions and performance is in a central place of biomimetics [1]. The term “biomimetic” was coined in the 1960s and is derived from the Greek words “bios” (nature) and “mimesis” (imitate) (imitation) [2]. Due to the increasing number of people who get injured and sick every year, the demand for medical devices that can help treat and cure these conditions has become very high. In the US, over 8 million surgeries per year are performed annually for these patients. Each year, over 100,000 people die while waiting for an organ transplant. The global market for bio-based medical devices was valued at USD 35.5 billion dollars in 2020. It is expected to reach USD 47.5 billion dollars by 2025 at a compound annual growth rate of 6.0% [3]. The complexity of biological systems has been optimized through the years to enable them to adapt to changes in their environment [4, 5]. For instance, Nacre and bone have incredibly tough and resilient characteristics. They can endure different kinds of impacts much beyond their constituent parts [6]. The feet of a gecko may produce enough adhesion to sustain many times its body weight, allowing it to climb walls and move quickly across ceilings [7]. Despite growing in a muddy area, Lotus leaves have great water repellence and appear to never get dirty [8]. Peacock feathers and Morpho butterfly wings have vivid and iridescent hues attributable to components that evolved independently of pigments in other species yet are akin to pigments in other species. There is a rising effort to discover techniques to synthesize composites with these extraordinary features mirroring those observed in nature, in addition to research activities committed to further understanding the causes underlying these interesting structural properties in nature. Understanding the design and properties of nature is also important to developing materials that are like those found in nature. Various techniques such as chemical vapor deposition, lithography, and self-assembly can be used to create advanced materials that mimic the properties of nature [9, 10].

CNTs in Biomimetic/Bioinspired Systems  259

11.2 Naturally Occurring Materials 11.2.1 Nacre and Bone The nacre, which is a typical shell or bone structure, is often used to demonstrate how natural composite materials can achieve exceptional properties. Its laminated composite structure is made up of aligned ceramic platelets and a small fraction of polymer. The nacre has a “brick and mortar” microstructure [11]. The mortar is made up of various proteins and chitin, and it is approximately 20 to 50 nm thick. The bricks are made up of 0.5 µm thick and 10–20µm wide aragonite (CaCO3) [12]. The former provides strength while the latter provides ductility. The main components of mortar’s toughening mechanism include crack bridging, aragonite-plate sliding, and energy dissipation [13]. The properties of these materials have led to the development of composite materials that have improved fracture toughness and strength–toughness balance.

11.2.2 Petal Effect and Gecko Feet There are two types of natural surfaces that have ultrahigh adhesion. The first is the petal effect, which allows plants i.e., roses to keep their petals in a spherical shape even when turned upside down (Figure 11.1) [14]. (a)

(b)

10 µm

(c)

1 µm

(d)

Figure 11.1  SEM images of the surface of a red rose petal (a, b). Shape of a water droplet on the petal’s surface (c) and when it is turned upside down (d). Republished from Ref. [14] with permission from the American Chemical Society.

260  Functionalized CNT for Biomedical Applications The second one is animals like the gecko lizards can cling to a variety of surfaces, such as moist, large, small, smooth, or rough ones. These creatures’ adhesive force can hold far more than their own weight [15]. A recent study revealed that the strong adhesion of the petals and feet of the geckos can be attributed to their periodic arrays of micro-and nanostructures. Red rose petals with nanofolds on the top are made with micropapillae. The gecko feet are made up of hundreds of tiny spatula-like structures having a diameter of 200-500 nm that are arranged into a series of wellaligned keratinous hairs which are known as setae and are 30–130 µm in length and 5 µm in diameter. These spatulas bend when they encounter any surface and interact with the surface through van der Waals forces which allows the gecko to climb up walls or across the ceiling.

11.2.3 Lotus Effect The leaves of plants have a unique wettability or water repellence that prevents water droplets from adhering to their surfaces. This phenomenon, which is known as the lotus effect, can cause the leaves to bounce off and leave behind dirt particles. This property is known as the “lotus effect” or “self-cleaning” property [16]. Studies suggest that the layer of (a)

(b)

100 µm

(c)

2 µm

Figure 11.2  Images of superhydrophobic lotus leaf (inset image is a water droplet on the surface of a leaf) (a). Low magnification SEM image of the lotus leaf surface structure (b). High-resolution SEM image of a single papilla, consisting of cilium-like nanostructures (c). Republished from Ref. [20] with permission from the American Institute of Physics.

CNTs in Biomimetic/Bioinspired Systems  261 epicuticular wax on top of randomly scattered micro-papillae contributes to the non-wetting properties of the papillae (Figure 11.2) and low surface energy epicuticular wax covering on these papillae are responsible for the non-wetting property [17]. The surface area of the liquid-solid contact rises with roughness, resulting in a larger energy barrier. Additionally, the rough surface retains air beneath the water droplet, forcing it to sit partially on an air layer. All these characteristics combine to create a superhydrophobic surface (contact angle > 150° and sliding angle 5°) [18, 19].

11.2.4 Structural Colors, Antireflection, and Light Collection A photonic structure is a type of optical structure that affects the flow of light. It can produce various optical effects such as antireflection and structural color and light collection. Many birds, insects, fish have structures with optical effects that allow them to change their appearance through the viewing angle. These structural colors can be used to attract mates or scare away predators. For instance, some insects and birds, such as the butterfly wings and the beetle scales, have structures with optical effects that allow them to change their appearance through the viewing angle [21]. Antireflective coatings allow insects to capture more photons, which helps them see better in low-light situations. Antireflection can also be used as a form of camouflage [22]. Brittle stars use photonic elements formed of calcite to capture light, allowing them to detect shadows and flee predators into dark crevices. The appeal of these structural colors has prompted a spate of attempts to duplicate them. If successfully manufactured, such structures might be used as sensing Bragg reflectors, high-resolution optical filters, and a replacement for pigment-based coatings [23].

11.3 Bioinspired Functionalized CNTs Material One-dimensional CNTs are made by a rolling sheet of graphene, which is an advanced material that can be used for various applications. They can be categorized into two types based on the number of graphene sheets used: single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). MWCNTs also contain double-walled CNTs. These materials are used in the production of structural components for aerospace and automotive applications due to their outstanding strength and flexibility. CNT reinforced fibers are stiffer than steel and resistant to external wear & tear. They have high electrical conductivity which makes them ideal for use in conductive polymer composites. The global carbon nanotube market

262  Functionalized CNT for Biomedical Applications was valued at $15.3 billion by 2017 and is expected to reach $130.2 billion by 2030, growing at a CAGR of 16.3% from 2021 to 2030. The increasing demand for CNTs in various applications such as medical devices and aerospace and defense and coatings is driving the market [24]. Since the beginning of the 21st century [25], CNTs are also expected to play a major role in the biomedical industry such as drug delivery, cancer therapy, tissue engineering, body implants, medical devices, and dental filling materials due to their various advantages, such as their small size, chemical stability, large surface area to mass ratio, and electronic polyaromatic structure. They are expected to be better drug carriers than the currently available drug carriers such as nanoparticles, polymers, niosomes, microparticles, and liposomes [25, 26]. They can be used as a drug delivery system by directly penetrating cells or by conjugating with various therapeutic molecules [25]. The medicine might be administered to the surface of the CNTs or put inside them. Both the medication and the CNT carrier enter the cells by endocytosis or insertion and diffusion in two different drug delivery conjugates using CNT systems [27, 28]. Surface attachment is ineffective when compared to internalization. In the former methodology, the conjugated drug-releasing pharmacological agents are degraded inside the cells, whereas in the latter method, the drug can be degraded in the physiological fluids before being internalized by the cells [29, 30]. CNTs were first binded to antineoplastic and antibacterial drugs for cancer and infection treatment, respectively, employing these cutting-edge medication delivery strategies. Although these properties are linked to highly desirable mechanical, electrical, and chemical properties for medical applications, CNTs’ biological applications are limited by their initial hydrophobic surface, which is prone to severe aggregation when exposed to aqueous solutions and has been found to be harmful to human health. Researchers, manufacturers, and those involved in the creation of medicinal infusions are also in danger [27]. To solve this issue in biomedical applications, carbon nanotube functionalization is essential. A chemical functionalization is a common approach for increasing CNT dispersibility, decreasing toxicity, and imparting desirable features in biological contexts [31]. After surface modification by targeting ligands and drug molecules, the resulting functionalized CNTs (f-CNTs) are less toxic [32] and immunogenic [33, 34], and show significant superiority as drug delivery devices capable of high drug payload and specific distribution into [35] biological systems [36, 37]. Functionalized CNT-based composites can replicate extracellular matrix (ECM) components and boost cellular connections and osteogenic potential by linking with different proteins and other macromolecules. The use

CNTs in Biomimetic/Bioinspired Systems  263 of osteogenic drugs or proteins is sometimes required for bone remodeling. By acting as a delivery channel for pharmaceuticals, growth hormones, proteins, and nucleic acids, carbon nanotubes with diverse functional groups can greatly influence the behavior and differentiation of osteoblasts and osteoclasts in bone tissue [38]. Due to 1) a mechanism of greatly increased fluid flow, CNT membranes offer an interesting chance to replicate natural protein channels, 2) the possibility to use ‘gatekeeper’ chemistry at the pores’ entry, 3) the ability to perform biochemical reactions on gatekeeper molecules, as well as 4) the ability to chemically functionalize each side of the membrane individually. Biomimetic nanomembranes are a subclass of synthetic nanomembranes that share some features with natural biological membranes, either partially or totally. The focus of this article is on recent advances in bioinspired hybrid materials made from functionalized carbon nanotubes. The following section will go through a few of the biomimetic materials research that has been published on the CNTs. Finally, many bioinspired hybrid material fabrication applications, current challenges, and future development are discussed. By pouring a mixture of gases at 750°C over SiO2/Si wafers, Qu et al. were able to build vertically aligned multi-walled carbon nanotube (VAMWNT) arrays [39]. The sheer force of the dry adhesives produced is about ten times that of a foot gecko. Due to the alignment of the carbon nanotube’s curly segments, strong binding was observed along the shear direction and facile lifting in the normal direction was also exhibited. Due to the higher packing densities and the better structure of conjugated carbon, a new type of single-walled carbon nanotube known as VA-SWNT could be produced. This new material exhibits strong van der Waals forces and a vertically aligned structure [40]. These nanotubes had a macroscopic normal adhesion force of up to 29 N cm-2. Due to the unusual thermal and electric properties of VA-SWNT, these dry adhesives exhibit reversible semiconducting behavior under load and good thermal resistance. The Ge et al. [41] were able to create a synthetic gecko tape. The tape was made with aligned CNT films and exhibited a high level of normal adhesion force of up to 29 N cm-2. The tape was made with VA-SWNT, which has mechanical and electric properties that are different from those of other commercially available nanotube films. These dry adhesives exhibit reversible semiconducting behavior under load and good thermal resistance. Majumder et al. used carbon nanotube-based membranes with “gatekeeper” chemistry to replicate the skin’s natural pores [42]. Carbon nanotubes were implanted into chelate of Ca2+ ions with sodium alginate (SA) and polyacrylic acid (PAA) by Wei et al. [43]. The prepared

264  Functionalized CNT for Biomedical Applications

Cutting

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Self-healing process of Ca-PAA-SA-CNTs hydrogels

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Ca-PAA-SA-CNTs hydrogel was further used to monitor human movements such as finger bending, knee bending, and breathing [43]. The produced hydrogel had excellent rheological features, such as stretchability, self-healing, and 3D printing. The self-healing property of Ca-PAASA-CNTs hydrogels was shown in Figure 11.3a. The resistive and capacitive

O

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Figure 11.3  (a) Cutting and self-healing processes of a circular Ca-PAA-SA-CNTs hydrogel and the corresponding self-healing processes with a diode used as an indicator. (b) Resistance and capacitance of Ca-PAA-SA-CNTs hydrogels before fracture and after self-healing. (c) Self-healing efficiency of Ca-PAA-SA-CNTs hydrogels quantitatively evaluated by calculating the recovery rate of G′ at strain sweeps from 1 to 1000% and back to 1% at a frequency of 1 Hz. (d) G′ and G″ of Ca-PAA-SA-CNTs hydrogels subjected to a strain sweep test with alternate small oscillation forces (1% strain) and large oscillation forces (500% strain). (e) Schematic illustration of the self-healing process. Republished from Ref. [43] with permission from American Chemical Society.

CNTs in Biomimetic/Bioinspired Systems  265 signals of the Ca-PAA-SA-CNTs hydrogels were characterized using an LCR meter (Figure 11.3b). Meanwhile, the rheometer’s strain sweep mode was utilized to quantify the hydrogel’s self-healing efficiency, which was obtained by calculating the recovery of storage modulus (G′). The hydrogel’s self-healing efficiency was assessed to be 86.6% during the process of altering the strain from 1% (G′ = 1051 Pa) to 1000% (G′ = 218 Pa) and then back to 1% (G′ = 910 Pa), as shown in Figure 11.3c. The hydrogel that was created was viscoelastic (Figure 11.3d). The improvement of the self-healing abilities of Ca-PAA-SA-CNTs hydrogels could be achieved by the chelation of Ca2+ and carboxyl group of PAA. The formation of an eggbox structure between Ca2+ and SA further improves the self-healing performances, as shown in Figure 11.3e. A study conducted by X Li et al. [44] discovered that multi-walled carbon nanotubes (MWNTs) and graphite (GP) compacts could stimulate the formation of bone in the dorsal musculature of ddy mice. The results indicated that the presence of MWNTs could stimulate the activation of inducible cells in soft tissues, which could then form inductive bone. Kuang et al. [45] constructed a peptide recognition element (PRE) (Figure 11.4) that may spontaneously coat SWNTs and give target selectivity. This method can be used to make SWNT-FET devices that can be tuned to detect traces of certain targets with PREs.

(a)

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Figure 11.4  Peptide P1ASP1C structure prediction using molecular dynamics simulations. Starting three different initial conformations a, b, and c (left panels) and equilibrated structures (right panels). Interaction between Trp17-Phe18 from one side and Tyr4-Trp6-Tyr5 from the other side stabilizes the sheet structure, while the bulkiness of Asn9- Asn10-Lys11-Thr12 destabilizes the helix structure. Republished from Ref. [45] with permission from American Chemical Society.

266  Functionalized CNT for Biomedical Applications In another study, the researchers [46] were able to produce highquality chitosan/carbon nanotubes/hydroxyapatite (CS/CNTs/Hap) composite materials with a high elastic modulus (509.9-1089.1 MPa) and high compressive strength (33.2-105.5 MPa). In vitro cell line tests on preosteoblast, MC3T3-E1 cells revealed that the composite materials can effectively stimulate osteoblast proliferation onto the surface of CS/CNTs/Hap. The authors proposed that the composites generated are suitable for bone tissue engineering, according to the research. A biomimetic scaffold made from chitosan, hydroxyapatite, and MWCNTs (CTS/MWCNT/nHAp) was created by mixing these components into an ice segregation-induced self-assembly approach [47]. They tested their scaffold’s efficiency by introducing multiple Mesenchymal Stem Cells (MSCs) derived from the periosteum. Because of their structural organization, surface properties, and cell survival, CTS/MWCNT/nHAp scaffolds can be used as a suitable material for bone tissue engineering. The hydroxyapatite component was then combined with carbon nanotubes and chitosan to improve its biological properties. The resulting biomimetic scaffold is then coated with an alpha-beta titanium alloy i.e., Ti-6Al-4V alloy to improve the biological and mechanical qualities of orthopedic material [48]. Another work [49] used glycerol phosphate to crosslink chitosan-grafted carbon nanotubes (MWCNTs and SWCNTs) and chitosan-grafted hydroxyapatite complexes. Using hydrogel nanofillers with a faster sol/gel transition increased the mechanical characteristics of the final material. Injectable composites of cross-linked carbon nanotubes and hydroxyapatite in thermosensitive gels showed promise, with good bioactivity, longer drug release, enhanced mechanical properties, and a faster gelation time. To improve the biocompatibility of a bone regeneration implant, a nanostructured CNTs-chitosan hybrid solution was created and electrophoretically deposited onto a titanium plate [50]. The effects of the CNTs-chitosan hybrid on the osteoblastic MC3T3-E1 cell line were investigated, and the results revealed that cell adhesion was stimulated. Furthermore, the nanotopographical property of the hybrid coating aided in the massive amount of protein adsorption and sustained release. Gholizadeh et al. [51] used MWCNTs, chitosan, and b-Glycerophosphate composites to make CNTschitosan composite scaffolds. The water absorption characteristics, porosity, and electrical conductivity of the composites were improved by increasing f-MWCNT to 1 w/v%. The compressive and tensile strength of the scaffold was also increased by adding up to 0.5% carbon nanotubes. The cell line results indicate that the composite obtained between 0.1 and 0.5 w/v% was appropriate for bone tissue engineering application because the functionalized MWCNTs make the nanotubes more biocompatible and soluble in

CNTs in Biomimetic/Bioinspired Systems  267 solvents, and the polymeric structure of the composites has not shown significant cytotoxicity, unlike other structured composites. In another work [52], the chitosan-MWCNTs scaffold was employed to investigate bone repair and regeneration. Using a water-soluble tetrazolium salt test and double staining techniques, the cytotoxic, apoptotic, and necrotic effects of MWCNTs-chitosan scaffold on chondrocyte cell lines were examined. The results suggest that the scaffold material had no effect on the viability of chondrocyte cell lines, implying that CNTs-chitosan composites are not toxic to cells and that the double staining method can distinguish between necrotic and apoptotic effects of chitosan-MWCNT nanocomposite on different cell lines. The scaffold’s mechanical capabilities are demonstrated by the stress-strain graph, and the inclusion of CNTs increased the elongation strength. Highly porous collagen composites functionalized with CNTs, chitosan, and HAp (Col/f-MWCNT/CS/HA) scaffolds were created using the freeze-drying method [53]. The Ca/P ratio of the reported Col/ f-MWCNT/CS/HA composite was 1.52, which is like the Ca/P ratio of natural bone (1.6). The -OH, -NH2, and -C=O groups of collagens have formed hydrogen connections with the -NH2 and -OH groups of chitosan, as well as the C=O and -OH groups of f-MWCNTs (Figure 11.5). To biomineralize the composites, a biomimetic technique using simulated body fluid was applied. The elastic modulus and compressive stress of the composite were measured before (523 and 37 kPa) and after mineralization (523 and 1112 and 57 kPa), respectively, to ensure structural unity during bone regeneration. The swelling (513.9-481.05%), porosity (98-95.7%), and contact angle (87.8°-76.7°) of Col/f-MWCNT/CS/HA were measured and compared to collagen, chitosan, and Col/f-MWCNT before and after biomineralization. Figure 11.6 shows how HAs were evenly distributed over the whole scaffold, with only minor aggregation. Sharmeen et al. [54] presented the results of their study on the properties of MWCNTs/gelation-chitosan composite materials functionalized with polyethylene glycol. They found that these materials have better thermal and mechanical properties, stiffness, wetness, and microfibrillar within pore walls which make them be used in the development of bone tissue engineering. Their improved mechanical and thermal properties can be utilized in porous gelatin/chitosan composite materials. An antibiotic drug, ciprofloxacin, was incorporated into a nanocomposite matrix. The antimicrobial susceptibility test and dissolution test were performed to evaluate the effects of MWCNTs on the drug’s release. The dissolution test (Figure 11.7) revealed that the drug’s release was initially sharp. However, it steadily decreased after being subjected to the antimicrobial susceptibility

268  Functionalized CNT for Biomedical Applications

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Figure 11.5  Biomineralization of collagen functionalized with CNTs, chitosan, and HAp composites using simulated body fluid. Republished from Ref. [53] with permission from Elsevier.

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Figure 11.6  Optical microscopy images of fabricated 3D (a) Col, (b) CS, (c) Col/­ f-MWCNT and (d) Col/f-MWCNT/CS scaffolds. Republished from Ref. [53] with permission from Elsevier.

CNTs in Biomimetic/Bioinspired Systems  269

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Figure 11.7  The release rate (%) of ciprofloxacin from different composite films at pH 7.4. Republished from Ref. [54] with permission from Elsevier.

test showing a sustained release. Brine shrimp lethality test revealed that the MWCNTs/gelatin-chitosan nanocomposites did not show any cytotoxicity. The combination of CNTs and sodium hyaluronate can help in the healing of tooth sockets faster in rats [55]. The researchers also found no effect of the HY-CNTs on the cardiovascular function of the rats on the direct ingestion even after 7 days. A second study revealed that the titanium surfaces coated with the sodium hyaluronate (HY)-functionalized CNTs bio-composites exhibited increased mRNA expression of various type I and III collagen, bone morphogenetic proteins 2 and 4, and osteocalcin, as well as mineralized bone nodule deposition [56, 57]. To repair bone in rat tibiae, CNTs were combined with hyaluronate (HY) bio composites. The bone defect was generated with a 1.6 mm diameter drill on the 7th and 14th days, and histological and morphometric examinations were performed. The ensuing histomorphometry demonstrated an enhanced percent with highly structured and denser bone trabeculae. Furthermore, increased levels of VEGF, collagen I (Col I), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) were seen in the Tibiae of an animal treated with HY-CNTs [58].

270  Functionalized CNT for Biomedical Applications The 3D poly(lactic acid) multiwalled carbon nanotube (PLA/ MWCNTs) nanocomposites scaffolds were created using a pressureactivated microsyringe microfabrication technique that allows for the direct fabrication of selected microstructures [59]. The intrinsic mechanical characteristics of the scaffolds can be varied by changing the ratio of CNTs to PLA, ranging from 60 to 170 MPa. The built scaffold demonstrated higher stiffness, porosity (60-75%), and cell survival (>75%) than a pure 3D microfabricated PLA scaffold on human fetal osteoblasts (hFOBs). Poly(d, L lactic acid) was added to vertically aligned-CNTs and hydroxyapatite in another study [60]. The in vivo investigation revealed that the scaffold induced bone remodeling. It was created using a poly(d, l-lactic acid)/vertically aligned CNTs/ hydroxyapatite bio-based scaffold by electrodeposition and immersion in the simulated physiological fluid. Human chondrocyte adhesion is aided by the produced material, which also reduces type I Collagen mRNA levels [61]. A 3D printed structure reinforced with polylactic acid/CNT filaments is created via melt extrusion [62]. After a 24-hour incubation period, the newly created composite filaments possessed mechanical properties and porosity comparable to pure polymer scaffolds, and biocompatibility was discovered on human MSCs (Mesenchymal Stem Cells). Photoacoustic (PA) differentiation of bone marrow-derived marrow stromal cells to osteoblasts was described by Sitharaman et al. [63] utilizing SWCNT-Poly(Lactic-Co-Glycolic Acid) (PLGA) films. Quantitative (alkaline phosphatase, calcium, and osteopontin) and qualitative (alizarin red stain) assays were used to assess cell differentiation. The osteodifferentiation of bone marrow-derived marrow stromal cells was improved by PA stimulation and SWCNTs in PLGA films, according to the study. Gupta et al. [64] used SWCNT and PLGA composites to explore the interactions between human bone marrow mesenchymal stem cells (BMSCs) and MC3T3-E1 cells in terms of cell proliferation, growth, mineralization, gene expression, and extracellular matrix synthesis. The findings revealed that the uniform distribution of SWCNTs on the PLGA matrix had no effect on the degradation rate. The biocompatibility of the composites was further demonstrated by the MC3T3-E1 and hBMSCs cells’ normal and non-stressed morphologies. Gupta et al. [65] confirmed in vivo biocompatibility of SWCNT/PLGA composite in a subcutaneous implant rat model. The consistent distribution of composite matches human trabecular bone in appearance, according to the

CNTs in Biomimetic/Bioinspired Systems  271 research. The composites’ potential was evaluated over a 12-week period, and the results revealed that PLGA degrades slowly. The composite material’s slow degradation rate may be advantageous for long-term investigations since the lower percentage of SWCNTs in the composite can considerably reduce implant toxicity. f-SWCNTs on the PLGA matrix had no effect on the degradation rate. The biocompatibility of the composites was further demonstrated by the MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (hBMSCs) normal and nonstressed morphologies. Pan et al. [66] employed a solvent evaporation method to create MWNTs-PCL scaffolds. These composite materials’ mechanical properties, such as tensile strength, swelling ratio, and degradation percentages, were discovered. Cell growth and differentiation were facilitated by the composites’ increased hydrophilicity and protein adsorption. As a result, the article demonstrates that PLGA-based carbonaceous composites can aid bone regeneration. The PCL-HA-CNT scaffold [67] was implanted in rat subcutaneous tissue for four weeks and generated soft fibrous tissues with neo-blood vessels on the scaffold’s three-dimensional holes without causing discomfort. A three-phased MWCNTs/nanohydroxyapatite/polycaprolactone composite [68] scaffold (mwCNT/nHA/PCL) exhibited excellent interfacial force and great miscibility and the scaffold has minimal porosity and delayed degradation, as well as big, crystalline hydroxyapatite in SBF solution. Furthermore, the scaffold enhances MG63 cell proliferation and differentiation. Later, the polymeric matrix of polycaprolactone-hydroxyapatite (PCL-HA) scaffolds loaded with CNTs were 3D printed to stimulate the proliferation of MG-63 cells [69]. The scaffold made up of 2% carbon nanotubes has outstanding mechanical and electrical conductivity. The scaffold’s compressive strength (4 MPa) was also compatible with trabecular bone. FloresCedillo et al. [70] used three different MWCNTs/PCL composites for bone regeneration: randomly dispersed MWCNTs/PCL,  aligned-MWCNTs/ PCL, and BGP-modified with aligned-MWCNTs/PCL. The mechanical and physicochemical properties of these composites were fascinating; for example, β-glycerol phosphate (BGP) modified MWCNTs/PCL had a tensile strength that was comparable to cancellous bone replacement (10-20 MPa). MWCNTs (0.3 wt%) did not produce toxicity in human dental pulp stem cells (HDPSCs), according to this study. Furthermore, BGP modified MWCNTs/PCL composites showed a greater proliferation of HDPSCs for up to 21 days.

272  Functionalized CNT for Biomedical Applications

11.4 Challenges and Solutions in Using CNTs 1. Due to their hydrophobic nature, CNTs are not ideal for the development of bio-compatible composite materials. Their poor dispersibility is a major impediment to the creation of such products. The strong non-polar covalent interactions and hydrophobic surfaces of sp2 hybrid carbon atoms in CNTs are responsible for their hydrophobicity. To improve their biocompatibility and aqueous solubility, continuous efforts should be made to functionalize biomolecules with CNTs. 2. Due to the unknown toxicity of carbon nanotubes, their usage as tissue engineering nanomaterials is of concern. Several studies have shown that their interaction with proteins, nucleic acids, and osteoblast-like human cells can increase cell affinity for various tissue engineering applications. Due to this, CNTs can cause inflammation on the surface of scaffolds and implants. This could result in loosening or migration of the desired implant location as well as the release of metal ions into the surrounding tissues. After CNTs are removed from implants and scaffolds, cross barriers can then distribute and become toxicological agents in the physiological environment. Functionalization can greatly boost bundled CNT dispersion and biocompatibility in an aqueous environment. New strategies and technologies are needed to overcome these issues. The surface of carbon nanotubes can be adjusted to prevent their negative effects either by adjusting the surface of carbon nanotubes or changing the manufacturing and purification procedures.

11.5 Conclusion and Perspectives The development of bio-inspired interfacial materials that mimic the natural functions of nature will have a significant impact on the energy and environment. Until now, developing a possible bioactive scaffold that resembles the extracellular matrix of genuine bone has been difficult. In the medical field, nanomaterial research and development have gained a lot of attention and demand. CNT-based composite materials outperform conventional biomaterials in terms of mechanical, electrical, and

CNTs in Biomimetic/Bioinspired Systems  273 chemical properties, making them a versatile platform in the biomedical field. CNT-based composites have become popular in tissue engineering. The enhanced physicochemical properties of carbon nanotubes have improved cellular interactions, adhesion, proliferation, and osteogenesis differentiation, resulting in more bone replacement and growth. Because of their large surface area, biocompatibility, and stimulation, CNTs’ activity as nanocarriers for drug administration and cellular transportation for bone disorders and deformities has been expanded. Even though CNTs have made significant advances in the field of bone regeneration and repair, there are still some hurdles that must be overcome through the collaborative efforts of researchers from around the world, from the laboratory to clinical application. Despite significant progress, the state of the art of CNT-based materials has not yet reached the maturity required for industrial applications in terms of scalability, stability, and reliability, which are complicated by their operating environments and the lack of simple strategies to exquisitely control local structural texture and chemical composition at multiple length scales.

Suggested Readings • Bio-Inspired Nanotechnology from Surface Analysis to Applications 2014, Eds. Marc R. Knecht, Tiffany R. Walsh, 9781461494461, Springer 2014. • Functional Properties of Bio-inspired Surfaces Characteri­ zation and Technological Applications 2009, Eds. Eduardo A. Favret, Néstor O. Fuentes, World Scientific, 9789812837028. • https://www.marketsandmarkets.com/Market-Reports/ biomaterials-393.html?gclid=Cj0KCQjw0PWRBhDKARIsAPKHFGi58CY12cNV0DDc-wK1c_iY4tt45QaU0xlbzTZXEbB2OP4I-7clS50aAkM-EALw_wcB • https://www.marketsandmarkets.com/Market-Reports/carbon-nanotubes-139.html • https://www.alliedmarketresearch.com/carbon-nanotubemarket • https://www.fortunebusinessinsights.com/industry-reports/ medical-devices-market-100085 • Functional Properties of Bio-inspired Surfaces Characteri­ zation and Technological Applications 2009.

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278  Functionalized CNT for Biomedical Applications nanotubes functionalized with sodium hyaluronate in rat Tibiae. Cells Tissues Organs, 204, 137, 2017. 59. Vozzi, G., Corallo, C., Daraio, C., Pressure-activated microsyringe composite scaffold of poly (L-lactic acid) and carbon nanotubes for bone tissue engineering. J. Appl. Polym. Sci., 129, 528, 2013. 60. Siqueira, I.A.W.B., Corat, M.A.F., das Cavalcanti, B.N., Neto, W.A.R., Martin, A.A., Bretas, R.E.S., Marciano, F.R., Lobo, A.O., In vitro and in vivo studies of novel poly(d, l-lactic acid), superhydrophilic carbon nanotubes, and nanohydroxyapatite scaffolds for bone regeneration. ACS Appl. Mater. Interfaces, 7, 9385, 2015. 61. Stocco, T.D., Antonioli, E., Elias, C., de, M.V., Rodrigues, B.V.M., de Brito Siqueira, I.A.W., Ferretti, M., Marciano, F.R., Lobo, A.O., Cell viability of porous poly(d, l-lactic acid)/vertically aligned carbon nanotubes/nanohydroxyapatite scaffolds for osteochondral tissue engineering. Materials, 12, 849, 2019. 62. Kim, H.-B., Patel, D.K., Seo, Y.-R., Lim, K.-T., 3D-Printed scaffolds with reinforced poly (lactic acid)/carbon nanotube filaments based on melt extrusion. J. Biosyst. Eng., 44, 120, 2019. 63. Sitharaman, B., Avti, P.K., Schaefer, K., Talukdar, Y., Longtin, J.P., A novel nanoparticle-enhanced photoacoustic stimulus for bone tissue engineering. Tissue Eng. A, 17, 1851, 2011. 64. Gupta, A., Woods, M.D., Illingworth, K.D., Niemeier, R., Schafer, I., Cady, C., Filip, P., El-Amin, S.F., Single walled carbon nanotube composites for bone tissue engineering. J. Orthop. Res., 31, 1374, 2013. 65. Gupta, A., Liberati, T.A., Verhulst, S.J., Main, B.J., Roberts, M.H., Potty, A.G.R., Pylawka, T.K., El-Amin III, S.F., Biocompatibility of single-walled carbon nanotube composites for bone regeneration. Bone Joint Res., 4, 70, 2015. 66. Pan, L., Pei, X., He, R., Wan, Q., Wang, J., Multiwall carbon nanotubes/polycaprolactone composites for bone tissue engineering application. Colloids Surf. B Biointerfaces, 93, 226, 2012. 67. Dorj, B., Won, J.-E., Kim, J.-H., Choi, S.-J., Shin, U.S., Kim, H.-W., Robocasting nanocomposite scaffolds of poly(caprolactone)/hydroxyapatite incorporating modified carbon nanotubes for hard tissue reconstruction. J. Biomed. Mater. Res. A, 101A, 1670, 2013. 68. Yang, H., Li, J., Liao, Q., Guo, H., Chen, H., Zhu, Y., Cai, M., Lv, M., In vitro evaluation of a novel multiwalled carbon nanotube/nanohydroxyapatite/ polycaprolactone composite for bone tissue engineering. J. Mater. Res., 34, 532, 2019. 69. Goncalves, E.M., Oliveira, F.J., Silva, R.F., Neto, M.A., Fernandes, M.H., Amaral, M., Vallet-Regı, M., Vila, M., Three-dimensional printed PCLhydroxyapatite scaffolds filled with CNTs for bone cell growth stimulation. J. Biomed. Mater. Res. B Appl. Biomater., 104B, 1210, 2016.

CNTs in Biomimetic/Bioinspired Systems  279 70. Flores-Cedillo, M.L., Alvarado-Estrada, K.N., Pozos-Guillen, A.J., MurguíaIbarra, J.S., Vidal, M.A., Cervantes-Uc, J.M., Rosales-Ibanez, R., CauichRodríguez, J.V., Multiwall carbon nanotubes/polycaprolactone scaffolds seeded with human dental pulp stem cells for bone tissue regeneration. J. Mater. Sci. Mater. Med., 27, 35, 2016.

12 Functionalized Carbon Nanotubes: Applications in Tissue Engineering Ajahar Khan*, Khalid A. Alamry† and Raed H. Althomali Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract

Tissue engineering is an arrangement of bioactive molecules, cells, and scaffolds for the reconstruction of injured tissues. Functionalized carbon nanotubes (CNTs) have emerged in the present time as pioneering components for the fabrication of the next-generation scaffolds to reconstruct injured tissue. Polymer-functionalized CNTs could exhibit various properties, including higher compatibility and ability of complexation with polymer matrix, improved dispersibility in different solvents, and responsivity to environmental stimuli. Furthermore, CNTs have broadly been investigated for their potential in tissue engineering because of their exceptional mechanical, physicochemical, and thermal features as well as suitable biocompatibility. CNTs based scaffolds have acquired significant recognition because of their brilliant attributes, which offer a synthetic but feasible microenvironment for chondrogenic differentiation, and cell proliferation. In this chapter, we aim to give an overview of the structural, physicochemical characteristics along with interactions and biodegradation of CNTs-based polymeric scaffolds with the biomolecules and outline their impacts on the extracellular matrix in the regeneration of neural, cardiac, bone, and cartilage tissue engineering. This chapter also provides a current state of information accessible investigating the utilization of the polymeric composites comprising CNTs and the development of the 3D scaffolds as well as the directions of future research and challenges. Keywords:  Tissue engineering, Functionalized carbon nanotubes, Cartilage regeneration, Polymeric scaffolds

*Corresponding author: [email protected] † Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (281–318) © 2023 Scrivener Publishing LLC

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12.1 Introduction Tissue engineering is an interdisciplinary area of science focusing on bioengineering, life science, pharmaceutical and materials science, and speculations of medicine that attempt to replace, reconstruct, and design damaged or diseased tissues/organs with biologic alternatives that can maintain and restore the regular function of the original organ [1–4]. Considerable benefits in the fields of tissues or organ transplantation, along with advancement in engineering and materials science, have aided in the ongoing advancement of regenerative medicine and tissue engineering. Particularly in the applications of tissue engineering, the evolution of nanomaterials is one of the promising research interests [5]. Furthermore, nanotechnology is emerging as a unique area of research that utilizes the excellent chemical and physical features of materials developed in the nano range, which are not accessible in bulk solids, therefore, it invoked great significance among worldwide scientific communities [6, 7]. Nanotechnology demands utilizing materials which acquire at least one physical measurement in 1-100 nm range to the fabricated systems, devices, and structures that have novel characteristics. The combination of tissue engineering and nanotechnology offers a broad platform for nano-materials in healthcare applications [8]. In the biomedical field, great interest and demands in the research of bone tissue engineering have been generated because of the various types of bone disorders witnessed at present time. Worldwide around ~20 million people are suffered from various diseases and accidents every year that involve bone disorders [9]. Furthermore, in recent times bone disorders such as rheumatic diseases, osteoporosis, infection, back pain, fracture, scoliosis, and tumors are the main concern because of the obesity, scarceness of regular physical exercise, and growing population [10]. Since the advancement of tissue engineering increasing there is a great demand for evaluating tools and improved monitoring of engineered tissues along with the development of newly hybrid biomaterials to assist tissue growth. Therefore, the preparation of promising bioactive material is a great requirement for tissue engineering that can mimic natural organs. Since the finding of carbon nanotubes (CNTs) by lijima [11], nanotechnology-­based on carbon has received great attention in comparison to the other nanoscale materials and appeared like a considerable material for several biomedical applications. They also possess biomechanical properties [12], good functionalization [13], high porosity [14, 15], followed by excellent tensile strength and thermal conductivity due to their nanostructure and strength of the bonds between carbon atoms. CNTs

CNT Applications in Tissue Engineering  283 have also acquired significant accomplishments in genetic engineering and might act as promising diagnostic tools and biosensors in biomedical research [16, 17]. The combination of polymer matrix with CNTs shows a variety of physicochemical and structural reinforcement properties, such as biocompatibility and flexibility, reduction of thrombosis, the introduction of angiogenesis, improvement of strength, and alteration of gene expression toward tissue engineering [18]. Typically, the length of the CNTs is in the range of micrometers and the diameters are in the nanometers range. It has been reported that the excellent structural morphology and aspect ratio (diameter and length) of CNTs affect their characteristics [19]. The theoretical surface area of single walled carbon nanotubes (SWNTs) (1, 315 m2/g) produces it as unique material for the applications of biomedical and nanorobotics. The inter distance amid each layer of multi-walled carbon nanotubes (MWNTs) is about 0.36 nm and the theoretical surface area of MWNTs is 100 m2/g [20, 21]. To replicate the morphology of fibrous proteins in the extracellular matrix, CNTs can be embedded within a hydrogel. According to previous reports CNTs had been used in hydrogel to improve the stiffness of the bulk gel [22, 23]. CNTs based hydrogels have been widely demonstrated for a wide range of applications in the human body, including brain circuit stimulation, skin growth, osteoblastic cell proliferation, and differentiation [12]. On the other side, CNTs exhibit such characteristics that make them a promising candidate for the future progress of tissue developments [24]. The important characteristic feature of CNTs in tissue regeneration is intrinsic morphology along with their anisotropic electrical and mechanical features. As anisotropic materials CNTs mimic the anisotropic alignment of cardiomyocytes present in the cardiac tissue by promoting the electroactive cardiac cells aligning in one direction. Moreover, the inherent CNT anisotropy might be united with synthetic approaches that permit the development of aligned morphologies to further gain the benefit of this feature. Therefore, several techniques including chemical vapor deposition [25], dielectrophoresis [26], or electrospinning [27], can be employed to design CNTs aligned cardiac constructs and encourage the improvement in the cellular alignment of cardiomyocytes. However, the utilization of CNTs incorporated scaffolds has not been reported so far for the application of promoting cell migration. Therefore, to evaluate the probability of utilizing CNT-based scaffolds as the synthetic extracellular matrix, their physical, mechanical, and biological properties need to be studied. In this review, we have overviewed investigations involving CNTs which are significant to the field of tissue engineering relative to their utilization on their own or in composition with suitable polymers.

284  Functionalized CNT for Biomedical Applications

12.2 Structural, Physical, and Chemical Properties CNTs are cylindrical carbon tubes assigning nanometer diameters and exhibit remarkable structural and electronic properties determine by length, diameter, and chirality. The carbon atoms in the backbone of CNTs present in a sequence of condensed benzene rings through a very large aspect ratio. As allotropes of carbon like graphene, CNTs exhibit the covalently bonded carbon atoms via sp2 hybridizations with one another. According to the cylindrical tubes numbers, CNTs might be comprised of a single cylinder-usually known as single-walled-CNT (SWNT), double-walled (two concentric cylinders), or multiple-concentric cylinders generally called multi-walled CNTs (MWNTs) [20]. SWNTs possessed hollow-long structures with one-atomthick walls, whereas MWNTs concentrically surrounded a central CNT and possessed multi-layers of graphitic carbon tubes. In general, the diameter of SWCNTs centered in the range of 1–2 nm, whereas for MWNTs centered around 10-100 nm having length differing from 50 nm to 1 cm [28, 29]. The structural morphology of SWCNTs involves sidewalls and tips. Three separate tubular structures can be identified via the orientation of rolling up a graphene film: chiral, armchair, and zigzag, that is designated with the chiral indices (m, n) [30]. The variations among diameter and chiral angle lead to different properties along with the different structures. The chiral and zigzag CNTs possess similar properties to semiconductors while armchair CNTs share analogous electrical properties to metals [31, 32]. SWNTs and MWNTs exhibited excellent mechanical strength, displaying tensile strength around 11-200 GPa and Young’s Modulus around 0.27–1.34 TPa [33–36]. CNTs also presented excellent thermal conductivity (5000 Wm-1 K) and superb electrical conductivity (104 S/cm2) [18, 37, 38]. The electrical properties and conductive structure of CNTs might be concluded through studying the electrical behavior of graphene [39, 40]. CNTs are linked through powerful nonpolar covalent bonds and their hydrophobic behavior on the surface produces them immiscible in the majority of organic solvents and water. Moreover, the high aspect ratio and Van de Waals forces of CNTs usually result in agglomeration [41]. Hence, the uniform dispersion of CNTs is a major challenge related to the CNTs utilization, deteriorating the mechanical, chemical, and electrical properties. To avoid this limitation, functionalizations of CNTs have been widely investigated. To functionalized CNT, two frequently used strategies are based on the covalent (chemical bond) and non-covalent (physical adsorption) functionalizations [42, 43]. The non-covalent approach is comparatively easy and can be carried out under mild conditions based on physical bonding because of diverse adsorption forces, i.e. π-π interactions,

CNT Applications in Tissue Engineering  285 hydrogen bonding, electrostatic force, and Van der Waals force [42–45]. The non-covalent functionalization techniques can be achieved in two ways: The first one is describes wrapping polymer chains or surfactants on the CNTs sidewalls and another approach can be accomplished via π-π stacking interactions between π-electrons of graphitic sheets on the CNTs surface and aromatic rings of grafted materials [32, 46]. The grafted polymer and surfactants can act as a dispersing agent to improve the solubility of CNTs in an aqueous medium [47, 48]. Noncovalent technique functionalized CNTs that possess high affinity towards biomolecules like enzymes, proteins, RNA, DNA, and peptides producing them attractive target delivery carriers to deliver biomolecules toward organs or cells [48–51]. On the other side, the covalent functionalization technique is focused on the preparation of covalent linkage among the main backbone of CNTs and functional groups. The defective carbon atoms at the end or on the sidewalls of both SWNTs and MWNTs might be oxidized through powerful oxidants (e.g. H2SO4/HNO3), generating carboxylated fractions or -COOH groups [32, 49]. To further enhance the activity of CNTs, permitting the linking of hydrophilic moieties, the -COOH are usually changed into acid chloride followed by amidation or esterification reaction [52–54]. In a study, Mooney et al. report the influence of the carboxylated-SWNT on human mesenchymal stem cells (hMSC) proliferation, viability, and differentiation [55]. It has been investigated that the COOH-functionalizedSWNT was easily dispersed in hMSC media and was least toxic to the cells. In Figure 12.1 (A) the green channel displayed that biotinylated COOHfunctionalized-SWNT were uptake through the hMSC and for the first 24  h primarily occupied a cytoplasmic location. A considerable amount of the labeled CNTs had assumed a nuclear location (Figure 12.1 Ac) after 6 days in culture. The hMSC did not show any evidence of green fluorescence that was not exposed to CNT (Figure 12.1 Ad). Furthermore, several molecules, such as biomolecules, metals, and synthetic polymers are found to be grafted to the carboxylated CNTs surface [56]. You et al. [57] reported that various types of aqueous soluble ionic polymer backbones, for example anionic (e.g. poly(acrylic acid), cationic (e.g. poly(2-(dimethylamino) ethylmethacrylate), and zwitterionic polymers (e.g. poly(3-(N-(3-methacrylamidopropyl)-N, N-dimethyl) ammoniopropane sulfonate)), were straightforwardly grafted to the MWCNTs surface through surface reversible addition-fragmentation chain transfer polymerization, assigning improve solubility. Redondo-Gomez et al. [58] studied the  modification of the MWNT surface using cholic acid through a chemical approach. The functionalized-MWNTs displayed facial bioamphiphilic nature, enhancing the stability of dispersion in water, low-polarity and organic polar solvents.

286  Functionalized CNT for Biomedical Applications (A)

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2 microns

2 microns

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Nucleus

Figure 12.1  (A) COOH-functionalized SWNT uptake via cell i.e. biotinylated CNT in the cell after (a) 24 h, (b) 48 h, and (c) 6 days and (d) hMSC alone (fluorescent images scale bar 130 μm). (B) (a) hMSC seeded on alginate and (b) human mesenchymal stem cells uncovered to carboxylated-SWNT and seeded on alginate for 24 h, (c) mitochondria, (d) cytoplasm and ribosomes on the endoplasmic reticulum, (e) and cell nucleus. Reprinted with permission from Ref. [55]. Copyright © 2008 American Chemical Society.

CNT Applications in Tissue Engineering  287 The covalent functionalization technique stabilizes the functionality and processability, being a robust and highly stable approach in comparison to the non-covalent functionalization technique [59].

12.3 Interactions and Biodegradation of CNTs with Biomolecule To enhance the biocompatibility of nanotubes, bioconjugation of CNTs is an appropriate approach to avoid possible health problems [60–62] followed by allowing the CNTs assembly into highly ordered structural form. To provide superb electronic and mechanical characteristics of CNTs to the microscale structures is of significant importance, which might be employed as active components [63]. As already studied for spherical polystyrene nanoparticles [64], CNTs can readily interact with a wide range of biomolecules (for instance proteins or lipids), creating a n ­ anoparticles-biointerface in biological environments [65]. This interface consists of thermodynamic and kinetics exchanges, and dynamic physico-chemical interactions between the surfaces of biological components and the surface of the n ­ ano-materials, such as proteins and phospholipids [66]. In nanoparticles-cell interactions the liquid/ solid interface is a significant task, which is generally not focused after entering the nanoparticles into a biological fluid (for example blood serum) and proteins compete for the surfaces of nanoparticles and lead to the construction of a protein corona [64]. Due to the definite ­physico-chemical characteristics of the nanoparticles, various progression can occur at this interface [66]. The gastro-intestinal tract (through digestion), blood vessels (through intravenous injection), the lungs (via inhalation), and the skin are the most possible way via which CNTs can penetrate into the human body [67], however, the pulmonary system (i.e. inhalation) is the main vital (human) exposure pathway for CNTs. Furthermore, CNTs can directly deposit onto the pulmonary surfactant layer positioned at the air/liquid interface of a lining layer of liquid casing the cells at the luminal side of the lung wall. This surfactant layer comprises mainly phospholipids (85-90%), with different proteins [68]. Furthermore, when deposited onto highly oriented pyrolytic graphite (HOPG) surfaces, adenine-­functionalized-SWNTs combined into well-­ordered structures like stripe [69]. In a similar approach, oxidized SWNTs were functionalized with derivatives of uracyl via amidation, emerging in toroidal structures (diameter ranges ~50 nm in), when deposited onto the surfaces of HOPG [70]. Sonication is a potent technique for dispersing bundled SWNTs in the aqueous solution of SWNTs and ribonucleic acid (RNA), double stranded deoxyribonucleic acid (dsDNA), and single

288  Functionalized CNT for Biomedical Applications stranded deoxyribonucleic acid (ssDNA) [71–73]. In the obtained dispersion, ssDNA helically wraps around the SWNT through π-π stacking of the aromatic nucleotide bases of the ssDNA. Furthermore, separating the surplus deoxyribonucleic acid (DNA) will neither influence the SWNTs dispersion nor be responsible for flocculation, demonstrating that the interaction of DNA-SWNT is comparatively strong. In addition, a report investigated that the phospholipids sited in pulmonary surfactant are capable to combine nonspecifically on the MWNTs backbone, [29] eventually stimulating the adsorption of blood plasma proteins [68, 74]. This prototype of adsorbed proteins is hypothesized to considerably affect the combination among biological systems and MWNTs as well as its effects. To understand this highly complex and dynamic interface further investigation has been accomplished. The hexagonal arrangement of carbon atoms of CNTs produced them to be highly biopersistent. SWNTs have been demonstrated to be biodegradable by natural enzymatic catalysis [75]. Allen et al. studied that, COOH-SWCNTs can be degraded by the use of the horseradish peroxidase (oxidative compounds) and hydrogen peroxide (the latter at low concentrations) at 48 °C in a cell-free environment after 16 weeks [76]. In a further investigation, it was studied that in a similar environment carboxylated SWNTs as well as pristine-SWNT both are biodegradable [76]. Although, whether peroxidase intermediates that are released within biofluids or human cells are involved in CNTs’ biodegradation is not presently understood. First of all, Kagan et al. investigated that reactive radical intermediates and hypochlorite generated from human neutrophils (the enzyme myeloperoxidase) are competent for SWNTs degradation in vitro (neutrophils and macrophages (lower degree)) [77]. The CNTs’ biodegradation inferred an innovative side of controlling the CNTs’ interaction with biomolecules. The comprehension of the bio/ nano interface is very essential and needs further, in detail study, as such interaction may conclude to a huge degree whether a nanomaterial is bio-­ compatible, bio-available, or might probably draw unfavorable influences on human fitness. To accomplish this understanding, new concepts, ideas, and analytical techniques are required.

12.4 Bio-Security of CNT-Based Scaffolds Toward In Vivo Analyses To enhance the mechanical strengths of polymers employed in bone regeneration, it has been proposed to introduce CNTs. Though, the concern arises related to the bio-security of the CNTs in vivo. CNTs are not considered biodegradable materials like some synthetic polymers or collagen. In contrast,

CNT Applications in Tissue Engineering  289 as a scaffold material CNTs will migrate to other body organs and remain in bone. Moreover, the biodegradability of the used scaffold material in tissue engineering is not a key parameter. Correspondingly, for materials that consist of inadequate biodegradability, there is a chance of inflammatory reactions that can occur from the degradation or remnants’ of by-products [78, 79]. Therefore, low degradability may essentially be a benefit for materials-­ based scaffolds in tissue engineering [80]. The strength and flexibility of the CNTs can positively contribute to the excellence of the engineered tissue. According to the literature survey, only a few studies of in vivo investigations have been carried out to evaluate the biocompatibility and propriety of scaffolds fabricated using CNT [81, 82]. Bhattacharya et al. studied the influence of a layer-by-layer assembled CNT-composite in vivo on bone tissue of rats and osteoblasts  in vitro [83].  The fabrication of composite was carried out using polyethyleneimine and SWNTs on polycaprolactone films [83]. In vitro analyses suggested that the cell differentiation of osteoblasts and matrix mineralization were enhanced on the CNT-based composites in comparison to the pristine titanium implants. Further, In vivo analyses suggested that the fabricated CNT-based composites stimulated bone repair and bone formation without inflammation or rejection signs after implanting in critical-sized rat calvarial defects. Lewitus et al. reported a unique strategy for producing CNT-fibers (CNF) composed with the agarose (polysaccharide) [84]. The obtained results suggested that the obtained CNT-fibers were not only non-toxic but also biologically modifiable and biocompatible. Furthermore, agarose/CNT hybrid material is revealed to assist response and cell attachment both in vivo and in vitro and found promising material for applications of biointerfacing with the nervous system and neural tissue engineering. Zande et al. reported a magnetic resonance imaging method to analyze the CNTs’ distribution pattern following release from a degrading poly(lactic-co-­glycolic acid) scaffold in vivo [85]. Poly(lactic-co-glycolic acid) scaffold with incorporated gadolinium-­labeled SWNTs subcutaneously was received by five rats. The obtained results suggested that magnetic resonance imaging is a brilliant approach to analyze the biological fate of gadolinium-labeled single-walled CNTs. Kaisai et al. studied the influence of carbon nanohorns on the regeneration of bone and their potential application for guided tissue engineering [86]. They evaluated a new material based on polytetrafluoroethylene membranes with carbon nanohorns for hard-­tissue reconstruction in a rat calvarial model by implanting specimens and their possible application for guided bone regeneration. After implanting of two weeks, they noticed that the formation of fresh bone was enhanced under the polytetrafluoroethylene membrane with carbon nanohorns in comparison to the pristine polytetrafluoroethylene. It was found that the growth of

290  Functionalized CNT for Biomedical Applications new bone begins from the edge of the presented bone and propagates to the center of the defect. However, after implanting of 8 weeks, no major variation was observed in the amount of freshly prepared bone noticed amid the membranes and the emergence of macrophages was reduced. Therefore they suggested the biocompatibility of the carbon nanohorns that promote bone regeneration in the early stage in vivo with a small inflammatory response and found that the formation of new bone was observed to be linked with the occurrence of macrophages. Saito et al. demonstrated the bone-tissue compatibility of CNTs by implanting MWNTs in mouse skulls [87]. The pathologic investigation suggested that MWNTs had no effect on bone even in CNTs contact with it and did not produce powerful inflammatory reactions, indicating adequate bone-­tissue compatibility. To determine the effect on the curing of bone, MWNTs were then implanted in tibial defects shaped in mice. Histopathology analyses showed that MWNTs were noticed in the bone matrix as well as in bone marrow. In another report, they implanted a 3D web based on recombinant human bone morphogenetic protein 2 (rhBMP-2)/carbon fiber porous composition into the dorsal muscle pouches of mice [80]. The insertion of web leads to the formation of fresh ectopic bone and the values of the bone mineral content and bone mineral density were considerably more than those procured a collagen sheet. The interface between the bone matrix and carbon fibers suggests that the fibers are integrated directly into the bone matrix, demonstrating higher bone-tissue compatibility. It was also revealed that the rhBMP-2/carbon-­fiber web composite restore a critical-size bone injury within a short stretch of time. Abarrategi et al. studied the effect of MWNT/chitosan scaffolds after implanting with BMP-2 for a period of three weeks into the subcutaneous pockets of rats [88]. This research group noticed ectopic bone formation, considerable degradation of the scaffold, and the non-appearance of chronic inflammation throughout the complete period of implantation. For the first time, Facca et al. reported in vivo analysis of plasma-sprayed CNT-reinforced hydroxyapatite coating on Ti implants incorporated in rodents’ bone [89]. It was found that the growth of bone was normal around hydroxyapatite-CNT coated implants along with no cytotoxicity or unfavorable consequence of CNT addition on cells and tissues of bone. The addition of CNT convinces higher osseointegration as compared to hydroxyapatite. It was also observed that the elastic modulus of the fresh bone was corresponding with the composites’ modulus, which revealed that the hydroxyapatite and CNTs’ occurrence improved the mechanical compatibility of the implant with the bone and lowered the probability of implant failure or fracture. The construction of CNT-based 3D scaffolds having potential for tissue regeneration tested in vivo is summarized in Table 12.1.

CNT Applications in Tissue Engineering  291

Table 12.1  Summary of the reviewed CNT-scaffolds tested in vivo for biomedicine applications. S. no.

Material

Fabrication method

Application

In vivo implant

Ref.

1

Poly (d,  l-lactic acid)/ graphene/MWNT-oxides

Oxygen plasma etching

Bone regeneration

Tibia of rats

[90]

2

CNT- membrane

Electrochemically

Transdermal nicotine delivery

Skin of hairless guinea pig

[91]

3

MWNT/collagen

Freeze-casting

Bone regeneration

Ectopic bone (mouse)

[87]

4

Polycaprolactone fumarate-CNT

Photocrosslinking

Nerve regeneration applications

PC-12 cell 

[92]

5

CNT/Agarose Fibers

Electrospinning

Neural Tissue Engineering

Rat cerebral cortex

[84]

6

CNT-hydroxyapatite scaffold

Electrophoresis

Bone tissue engineering

Bone mesenchymal stem cell lines

[93]

7

poly(3, 4-ethylenedioxythiophene) (PEDOT)-CNT/sucrose scaffold

Vapor phase polymerization

Tissue engineering

C8-D1A astrocyte 

[94]

(Continued)

292  Functionalized CNT for Biomedical Applications

Table 12.1  Summary of the reviewed CNT-scaffolds tested in vivo for biomedicine applications. (Continued) S. no.

Material

Fabrication method

Application

In vivo implant

Ref.

8

CNT-Alginate gel

Gel formation/ freeze-casting

Tissue engineering

Subcutaneous (rat)

[95]

9

Poly(3-hydroxybutyrate)/ MWNT

Solvent casting and particulate leaching

Tissue engineering

Subcutaneous

[96]

10

Poly(propylene fumarate)/ SWNT

Particulate leaching

Tissue engineering

Femur condyle and subcutaneous (rabbit)

[97]

11

MWNT/collagen

Coating

Tissue engineering

Bone

[82]

12

CNT-fibers

-

Restoration of Myocardial Conduction

Myocardial

[98]

CNT Applications in Tissue Engineering  293

12.5 CNTs Towards the Bone Compatibility The compatibility of bone-tissue of CNTs and their effects on bone regeneration are significant concerns, although the CNTs’ consequences on bone have not been described. In a bone implantation testing report, it was observed that the CNT/alumina composite exhibited better compatibility of bone tissue and was directly associated with new bone. The in vitro cell attachment analysis was carried out for fibroblasts, chondrocytes, osteoblasts, and smooth muscle cells where the composite of CNT/alumina displayed cell attachment and proven bone tissue compatibility and good biocompatibility [99]. The discussed in vivo investigations suggested quite improved bone compatibility of carbon nanotubes, some in vitro experiments have displayed cytotoxic consequences for bone cells. Because of their good affinity with CNTs, nutrients in the culturing medium requisite for the growth of cells can combine with CNTs, lowering the cell proliferation rate [55]. It has been studied that CNTs may also bind to the receptors at the cellular level and on the cell membrane block the pores, or even perforate the whole cell membrane or physically rupture [100]. This behavior can also permit the gene or drug delivery into cells by means of CNTs (such as impalefection). After internalizing into the cells, the fabrication of reactive O2 species might be stimulated because of the increased surface reactivity and the high surface-to-volume ratio of CNTs. High concentrations of reactive oxygen species are generally linked with the spoil intracellular components through uncontrolled oxidation and can consequence in cell apoptosis [100–102]. Liu et al. studied the reserve proliferation, mineralization, and osteogenic differentiation of human mesenchymal stem cells on COOHCNTs [103]. In preference to the reactive oxygen species mechanism, they revealed that the articulation of adipocyte differentiation specific genes and osteoblast specific genes was significantly decreased in the occurrence of COOH-CNTs. The microscopic investigation suggested the connections of CNTs with proteins positioned in the cytoplasm or on the cell membrane. Therefore it was revealed that the toxicity arises from modulation during the Smad-dependent bone morphogenetic protein signaling approach [103]. The obtained data was in a good accord with the research of Mu et al. who reported the repression of bone morphogenetic protein signaling using COOH-SWNTs [104]. SWNTs are considered to be promising materials for controlling intracellular signaling cascades and regulating genetic regulation. Furthermore, in vitro experiments showed that carbon nanotubes have also revealed a dose-dependent influence toward gene expression in human fibroblasts [105]. In order to consider the adverse effects, several

294  Functionalized CNT for Biomedical Applications techniques have been carried out to reduce the cytotoxic issues in the last two decades, for instance by surface functionalization, purification, shape engineering, and compositing with suitable biocompatible materials [106, 107]. The metal impurities (such as catalysts) might be remaining as by product through the fabrication of CNTs. Such metal impurities can propel more cytotoxicity to bone cells [102]. The appearance of toxicity can possibly reduce via eliminating these impurities using acid oxidation and supporting the growth of cells [108]. Nimmagadda et al. studied the overall physical and chemical influence of different SWNT fabrications on in vitro cell metabolic activity and viability [109]. They described the 3T3 cells (mouse fibroblasts) behaviors of on 3 different SWNTs modified with glucosamine [109]. The obtained results showed that metabolic activity and cell viability 3T3 cells of were influenced by the preparation method and concentration of CNTs. Purification and functionalization of CNT surface to reduce hydrophobicity or to incorporate functional groups considerably mitigated the cytotoxicity [109]. Poly(ethylene glycol)ylation (PEGylation) is one of the significant approaches to improving bone biocompatibility. Sonicating of the COOHCNTs in the solution of dimethylformamide and treating with oxalyl chloride, along with the addition PEG under mechanical agitation at elevated temperature is a common method for fabricating hydrophilic PEGylated CNTs [110]. Nayak et al. fabricated a thin film based on PEGylated MWNTs and determined their capability to effects human mesenchymal stem cells’ morphology, differentiation, and proliferation into osteoblasts [111]. They reported that these functionalized nanotubes did not possess cytotoxicity. In addition, the viability of human mesenchymal stem cells was increased and the osteogenic differentiation of human mesenchymal stem cells was stimulated, with simultaneous bone matrix mineralization. It is significantly noted that the assay being employed to evaluate the CNTs’ cytotoxicity is also serious as other approaches may generate different readings and results [112]. Therefore it is essential to indicate the assay utilized when one investigates the cytotoxicity effects of CNTs.

12.6 Applications of Functionalized CNTs in Tissue Engineering 12.6.1 Functionalized CNTs for Cardiac Tissue Engineering Among the other carbon-nanomaterial based composites, CNTs attributed promising features that produce them as potential candidates for the fabrication of future cardiac implants [113]. Numerous types of research

CNT Applications in Tissue Engineering  295 investigations to encapsulate the carbon nanomaterials into cardiac tissue regenerations have targeted the utilization of graphene derivatives, gold nanoparticles, and CNTs based materials [114–117]. The incorporation of CNTs has permitted overcoming several drawbacks, offering patches with improved mechanical and conductive properties [113]. CNTs accelerated the maturation of cardiomyocytes and enhanced the functionality of cardiac tissue in comparison to other conductive carbon-based nanomaterials, for instance, reduced graphene oxide (GO) [118, 119]. Moreover, CNTs bound to or encapsulated into polymeric moieties act more similar to particulate materials rather than nanometric materials, accordingly no interaction with the cellular cytoskeleton, with no inflammation, cytotoxicity or any significant adverse effect when investigated in vitro or in vivo for the application of cardiac tissue engineering [120]. CNTs offered electrical signals to glutaraldehyde cross-linked gelatin hydrogels [121]. The 3dimonsional scaffolds stimulated electrical activity and cardiomyocyte contractile in vitro. The results appeared to be in vivo analysis of the patch implanted into rat infarcted hearts, noticing its fruitful biohybridization among the host myocardium and the successive cardiac tissue reconstruction [121]. The native myocardium extracellular matrix comprises a wide range of aligned nanofibrous-like and electrically conductive proteins, mostly laminin and collagen that facilitate the propagation of electrical signals alongside the cardiac cells to ultimately assist the synchronous contraction of the cardiac tissue. The fabrication of nanofibrous matrices with oriented morphologies to mimic biologically was found to promote the construction of aligned engineered cardiac tissue with mature phenotype and improved performance [122]. This type of orientation can lead to the aligned association of cardiac cells, gap junctions, and sarcomeres, to facilitate the electric proliferation and succeeding synchronized contraction. Additionally, microelectrodes using aligned-CNT at lower voltages can permit pacing in close association with cells, accordingly regulating the electrical pulses amid tissue and cells constructs followed by controlling their function and behavior. Such type of morphology, which attains close resemblance to the nanofibrous microstructure of the native extracellular matrix, allows localized stimulation along with guiding cardiac cells. Furthermore, Ahadian et al. [123] aligned CNTs in gelatin methacrylate through dielectrophoresis. In this finding, mouse  embryoid bodies  were cultured in the microwells developed on the scaffolds of aligned CNThydrogel. The utilization of CNT-hydrogel on mouse embryoid bodies upon electrical stimulation confirmed a better cardiac differentiation, in comparison to randomly-dispersed or nonconductive CNT hydrogel controls [123]. Wu et al. developed a 3D hybrid scaffold using an aligned

296  Functionalized CNT for Biomedical Applications (a)

4th 3rd 2nd 1st

Epicardium

Endocardium

Epicardium

Multi-layers NFYs-NETs

A gradual transition of cellular alignment

Hydrogel precursor solution UV light

(c)

A gradual transition of alignment

(b)

Endocardium

(e)

(d)

Photocrosslinking

2-layer NFYs-NET

500 µm

2-layer 3D scaffold

1st layer (f)

2nd layer (h)

(g)

300 µm

Cells within 2-layer 3D scaffold

(k) Fraction of Cells

3D view (j)

500 µm

F-actin/DAPI

0.3

300 µm

2-layer 3D scaffold

0.2 0.1 0.0 -90° 0° 90° Angle of cells direction

300 µm

(l)

60

90

30 0 330 300

120 150 180

270 (-90)

210 240

Figure 12.2  Fabrication of 2-layer 3D aligned conductive nanofiber yarns network (NFYsNET)/Gel hybrid scaffolds and cardiomyocytes cultivation. (a) Schematic representation of myocardium representing a steady transition of aligned cell layers from endocardium toward epicardium. (b) Schematic representation of NFYs-NETs assembled multiple layers with the steady transition of orientation. (c) The possible approach of 2-layer 3D scaffold construction through incorporating two layers of NFYs-NET with orthogonal adaptation after photo-cross-linking in methacrylated gelatin hydrogel shell. The gross image (d) and 3D representation of confocal image (e) of 2-layer 3D scaffolds, where hydrogel was stained with green and aligned conductive nanofiber yarns network was stained with red, respectively. (f) The representation of cells culturing on two aligned conductive nanofiber yarns network layers in shell of hydrogel. Fluorescent images of cardiomyocytes through staining F-actin (green) on the scheme layer with horizontal (g) and vertical direction (h) in the fabricated scaffold. The top view (i) and 3D view (j) of confocal images of cells through staining F-actin/ DAPI. The quantitative distribution investigation of cellular orientation verified that cells were aligned on each layer while were perpendicular on different layers (k, l). Reprinted with permission from Ref. [124]. Copyright© 2017 American Chemical Society.

CNT Applications in Tissue Engineering  297 conductive nanofiber yarns network (combination: CNT, silk fibroin, and polycaprolactoned) via weaving in a hydrogel shell for mimicking the native cardiac tissue structure. The fabricated nanofiber yarns network was demonstrated a huge probability for constructing 3D cardiac anisotropy towards cardiac tissue engineering [124]. The incubation of cardiomyocytes in the nanofiber yarns network scaffolds verified their improved biocompatibility and aptitude to assist cellular elongation and alignment, improving cardiomyocytes’ function and maturation (Figure 12.2).

12.6.2 Functionalized CNTs for Neuronal Tissue Regeneration Nerve damage/injury can happen through virus infection, hypoglycemia, anoxia, diabetes, and external trauma [18]. However nerve axons are competent to regenerate, the size of the damage is restricted to 5 mm and adjustment is necessarily required to surgically fix the injured nerve ends. Hence, numerous types of nerve therapies have been investigated such as nerve autograft, coaptation, nerve conduit, and nerve allograft [125]. An approach which is utilized to fix the injured central nervous system cannot be left conveying the axons regrowth, the regeneration of neurons, and the plastic recasting of neuronal circuitry which are emerged from stem cells [126–128]. Although this is a very complicated matter, the growth of axons requires overcoming inhibited and unfavorable environments and needs target recognition, the rebuild of functional synapses, and suitable axonal spatial organization. Stem cells are able to differentiate into a specific neuronal lineage and need to be alive [18]. In this view, the artificial implants based on synthetic material, for instance, 3D scaffolds are needed to offer a bioactive and biocompatible atmosphere, permitting the maintenance, differentiation, and attachment of nerve cells and ultimately involving the construction of functional neuronal assembly [127, 129, 130]. Additionally, the surface offered for the regeneration of the nerve requires having suitable dimensions to limit any nerve compression and damage and to permit the discharge of neurotrophic factors and diffusion using the proximal site of axon damage [131]. To permit in vivo manipulation flexibility is essential, requiring proper elastic modulus and ultimate tensile strength (0.58 and 1.4 MPa respectively for decellularized nerve tissue) [132–134]. In due course, nanotopography possesses huge significance as the neurons’ contact with their growing substitutes arises at the nanoscale level for example neural cell adhesion molecules-NCAM, integrins, and N-cadherin which is very delicate to the substrates nanoatmosphere [135, 136]. Several kinds of research have been studied the utilization of CNTs directly as a platform for neuronal tissue regeneration and outcomes suggested that carbon

298  Functionalized CNT for Biomedical Applications nanotubes are competent to assist neurons attachment, ease the generation of more elaborate and longer neurite as well as promote cell differentiation [137–139]. The combination of CNTs with surprising mechanical properties and high electrical conductivity provides a route for non-conductive materials towards neuronal tissue regeneration along with minimizing the toxicity behavior of CNTs. Due to these properties; several reports have demonstrated the composition of biocompatible polymers with CNTs to fabricate 2D electrical conductive membranes or meshes for neural tissue regeneration. Shao et al. [140] studied a unique approach to fabricating a membrane that comprises PDDA (poly(dimethyldiallylammonium chloride) and CNTs through a layer-­by-layer congregation. Fascinatingly, outcomes suggested that the negatively charged CNTs and positively and negatively charged PDDA can show more homogeneous CNTs dispersion in the polymer backbones. The prepared substrates generated an effective regulatory signal for neural stem cells with respect to adhesion, outgrowth, differentiation, proliferation, and electrophysiological advancement of neural stem cells (NSCs) derived neurons. Furthermore, they suggested a plausible method that the integrin-mediated contacts among NSCs and substrates initiated focal adhesion kinases (FAK), leading to activation of a progression of signaling for example a wingless-­related integration site (Wnt) signaling pathway which controls NSCs proliferation, mitogen-­ activated protein kinase (MAPK) signaling route which controls differentiation of neural stem cells, and a phosphatidylinositol 3-kinase and protein kinase B (PI3K-AKT) signaling pathway to controls the cell survival [140]. Vicentini et al. [141] reported  3 types of organic-­functionalized carbon nanostructures namely, reduced-graphene oxide (GO), carbon nanohorns, and  p-methoxyphenyl functionalized-MWNTs, employed as nanofillers for the fabrication of well-dispersed and homogeneous composites of poly(l-lactic acid), a biodegradable and biocompatible U.S. food and drug administration (FDA)-approved polymer. A 2D film was fabricated by all these composites through electrospinning for neural tissue regeneration. The MWNT containing material possessed higher elongation break and Young’s modulus and but reduced electrical resistance. These fabricated scaffolds demonstrated improved biocompatibility and assist the proliferation of SH-SY5Y (human neuronal precursor cell line).

12.6.3 Functionalized CNT for Cartilage Tissue Engineering Biocompatible and eco-friendly polymers are broadly utilized in the construction of scaffolds for cartilage tissue regeneration. Biopolymers have good rates of degradation but possess lower mechanical strengths [142].

CNT Applications in Tissue Engineering  299 In this regard, synthetic polymers with high mechanical stability such as polyglycolic acid (PGA), poly (lactide-co-glycolide) (PLGA), polylactic acid (PLA), and polycaprolactone (PCL), have been evaluated for regenerative medicine and tissue engineering. A composition of biocompatible materials such as CNT and graphene oxide (GO) with a suitable polymeric matrix can advance the biomechanical characteristics of the polymeric matrix. In a report, PLGA and COOH-MWNTs based nanocomposite film was found to be enhanced the biocompatibility of osteoblasts and proved to be a suitable implant substrate for tissue engineering [143]. This report confirmed the cellular reactions of osteoblasts grown on the fabricated film. The experimental outcomes suggested that interaction with the nanocomposite films modulated osteogenic differentiation in osteoblasts, stimulated DNA synthesis and S phase accumulation, and improved cell proliferation [143]. According to the literature, chondrocytes was cultured in composite mixtures of agarose (2%) and two preparations of nanotubes, i.e. covalent polyethylene glycol (PEG)functionalized SWNTs, and SWNT-COOH [144]. The carboxyl functionalized SWCNT displayed better viability than a control scaffold, whereas the scaffold based on SWCNT-PEG displayed reserved proliferation. Fascinatingly, a larger amount of SWCNT-PEG regardless of the lowest cell viability accomplished more advanced extracellular matrix (ECM) production. Karbasi et al. fabricated Poly(3-hydroxybutyrate) (PHB)/ chitosan scaffolds comprising MWNTs through electrospinning for cartilage tissue engineering [145]. The obtained results verified that mechanical strengths are approximately similar to characteristics of cartilage. Therefore, PHB-chitosan/MWNTs electrospun scaffold possessed appropriate mechanical and structural behaviors which can be employed for cartilage tissue regeneration [145]. In a study, Zadehnajar et al. prepared PCL/gelatin based scaffold with 1 wt.% of MWNTs using the electrospinning technique [146]. It was found that the bioactivity and hydrophilicity of the PCL/gelatin/MWNTs were increased due to MWNTs. The mechanical stability of the PCL-gel/MWNTs scaffold was increased to 2.92 MPa in comparison to the PCL/geletin because of the presence of 1 wt% of MWNTs. The weight loss of the PCL/geletin scaffold after 14 days was more in comparison to the PCL-gel/MWNTs scaffold. The PCL/ geletin scaffold was completely degraded within 32 days due to the fragile interaction amid the polymers. The rate of degradation of PCL/geletin/ MWNTs scaffold was reduced because of the ionic bonds among the carboxyl group of MWNTs and amine group of geletin which enhanced the chemical strengthen of its structure. According to the methylthiazolyldiphenyl-tetrazolium bromide (MTT) investigation, both electrospun

300  Functionalized CNT for Biomedical Applications scaffolds were cytocompatible for up to seven days [146]. Furthermore, the poly 3-hydroxybutyrate-chitosan-1 wt% MWNTs functionalized by COOH (MWNTs)/silk nano-micro scaffolds were developed via electrospinning for cartilage tissue engineering [147]. Chitosan, MWNTs, and silk promoted the attachment and development of chondrocytes. The carboxyl groups in MWNTs simulated body fluid, facilitating the deposition of phosphorous and calcium that can behave similarly to ceramic reinforcement and improved bioactivity of the scaffold. Additionally, scaffold comprising 1 wt% MWNTs attributed enhanced tensile strength (46.68 ± 2.20 MPa) that might be appropriate for cartilage tissue engineering. The calcium concentrations in the scaffold comprising MWNTs were improved from 2.20 wt% to 3.73 wt% after 28 days, due to adsorption of phosphate with the amine group of chitosan and depositions of Ca2+ with carboxyl groups of MWNTs. Besides the MWNTs degrade in about 56 days because of the ionic bond among carboxyl and amine groups in poly 3-­hydroxybutyrate and MWNTs, it can properly serve for long-term cartilage tissue engineering [147].

12.6.4 CNT for Bone Tissue Regeneration Bone is a type of connective tissue which is a vascular and extremely specialized form and found to be very promising towards maintaining the skeleton shape. During movement defending the soft tissues in cranial, transferring the forces of muscular contraction, pelvic and thoracic cavities, regulating the ECM, and maintaining ions, blood pH, and blood production [148]. Bone has the ability to regenerate and heal itself in the case of partial fracture, or damage. However, bone is unable to heal itself in pathological fractures, primary tumor resection or traumatic bone loss, when injuries surpass a critical size (5 mm) [149]. Therefore, various types of therapies were evaluated such as xenografts, allografts, and autografts but these approaches display different drawbacks for example donor site morbidity and limited supply for autografts, risk of diseases transmission and rejection for allografts, and poor clinical outcome and risk of immunogenicity for xenografts [150–152]. As a result, the utilization of synthetic bone 3D scaffolds, that afford the essential reinforcement for cell attachment, differentiation, and proliferation are requisite. Literature survey reports that CNTs block displayed strong alkaline phosphatase (ALP) action with the occurrence of recombinant human  bone morphogenetic proteins (BMP-2), representing better osteogenesis activities. Tanaka et al. [153] studied the capability of the 3D porous CNT scaffold for bone regenerative medicine and comparison of CNTs was also carried

CNT Applications in Tissue Engineering  301 out with hydroxyapatite in vivo and in vitro. The experimental outcomes suggested that CNTs displayed improved absorption and release capability of protein. In vivo analyses indicated that porous morphology of CNT had better osteoconduction, greater proliferation of cell and higher bone generation upon incorporation with recombinant human BMP-2 [153]. Tanaka et al. [154] also reported the preparation CNTs based 3D block structure and demonstrated the efficacy of CNTs’ as scaffold materials for bone tissue engineering and is a comparison with PET-reinforced collagen. They found that the prepared scaffold displayed considerable variations with the femoral bone of rat with analogous compressive strength of 62.1 and 61.86 Mpa respectively. It was also found that CNT scaffolds reported prior cell adhesion than PET-reinforced collagen scaffolds. Khalid et al. [155] produced MWNT/hydroxyapatite scaffolds using different loadings of CNTs (1, 3, and 5 wt%). Investigations through human osteoblast sarcoma cell lines indicated the dose-dependent cytotoxicity of the fabricated scaffolds and the viability of cells reduced with the rise in content of CNT. Oyefusi et al. [156] produced promising bone implant materials by successfully grafting hydroxyapatite onto graphene nanosheets and CNTs. These materials were further examined in terms of cell differentiation and proliferation rate of the temperature-sensitive human fetal osteoblastic cell lines. The obtained outcomes verified that both CNTs-hydroxyapatite and graphene-hydroxyapatite felicitated cell differentiation and growth. In another report, Gupta et al. [157] developed composite microspheres based on ­polylactic-co-glycolic acid and SWNT for bone tissue regeneration. The considered MC3T3-E1 cells (MC3T3-E1 subclone 4, precursor osteoblasts) were investigated in terms of gene expression, proliferation, and adhesion. They reported that MC3T3-E1 cells adhered, exhibited normal, and grew, the addition of SWNT and non-stressed morphology strengthened gene expression and rate of cell proliferation in comparison to pure polylactic-co-glycolic acid scaffolds [157]. Facca et al. report in vivo analyses of implants based on plasma-sprayed-CNT-reinforced-hydroxyapatite (HA) coating on Ti incorporated in rodents’ bone [89]. They noticed normal bone growth in the vicinity of HA-CNT implants and found that the presence of CNT stimulates larger osseointegration in comparison to HA. For this during one month Ti rods or beads with 3 different coatings: (i) uncoated Ti (group 1), (ii) HA coating (group 2), and (iii) HA/4 wt % CNT (group 3) coating were implanted in the external condyle of rodents’ bone (Figure 12.3(I-a)). They found that after one month, for all groups the cortical defect was restored because of the development of the neocortical bone Figure 12.3(II). As shown in Figure 12.3(III) with toluidine blue coloration, the gray layer (HA-CNT coating) was observed to be firmly connected

302  Functionalized CNT for Biomedical Applications (a)

(b)

Implant inside femoral condyle

Femoral head

Implantation site

I (a) group 1 C

(a) group 2

OSTEOCYTE

C

II

(a) group 3 C COATED LAYER

TRABECULAR BONE

(b) group 1

(b) group 2

(b) group 3

C

C

C

COATED LAYER

COATED LAYER

TRABECULAR BONE

III Figure 12.3  (I)(a) A rod incorporated inside the femoral condyle (through a bony defect). (b) femoral bone of rat got from group 3: HA-CNT-coated Ti implant. (II) Experimental outcomes with rod-shaped implants for rat bones (histological data 40×). (a) Mallory coloration views and (b) hematoxylin-eosine coloration view thick trabecular, normal, and hematopoietic marrowbones, without any inflammatory tissues and reactions. A thicker for group 3 (HA-CNT coating) and attached to newly grown bone tissues closed to the cavity (c) caused by the retrieved implant. (III)n for group 3 implant with toluidine blue coloration(histological image (5×)) displaying HA-CNT coating firmly connected to freshly developed bone tissues and near to the coating normal neobonealong with normal cell morphology. Reprinted with permission from Ref. [89]. Copyright © 2011 American Chemical Society.

CNT Applications in Tissue Engineering  303 to the freshly developed bone tissues. According to this study in vivo analysis verified that for the HA composite CNTs as nanoreinforcement can be employed for applications of orthopedic without any adverse effect [89].

12.7 Future Perspectives and Challenges The fabrication of new effective nanomaterials and their utilization in tissue engineering is really important for the regrowth or repair of damaged or injured tissue. To meet this demand several researchers try to construct new biomaterials along with the existing nanotechnology, through different compositions of various nanomaterials. The construction of scaffold materials proficient in generating preferred tissue and cellular behavior is greatly favored in the area of tissue regeneration. Several reports have suggested that due to their exceptional properties, CNTs are potential candidates in fabricating such materials with finely tunable and improved ­physico-chemical and mechanical characteristics. A variety of multifunctional hybrids with desired structures have been demonstrated to be efficient in improving the adhesion, directing the differentiation of bone-related cells, and promoting proliferation, along with offering appropriate sites for the deposition of minerals. Furthermore, pretty desirable in vivo bone biocompatibility has also been noticed after implanting these materials inside animals. Despite having too many reports, progress in this field is still in its beginning and a lot of research is required to completely investigate the limitations and potential of CNTs based materials in tissue engineering. The directions of future research along with the challenges that need to be addressed are as follows: (i) Advanced structural morphologies along with the 3D porous structure that replicates the structure of the original bone are needed. These sophisticated morphologies not only produce mechanical hold against the in vivo actions but also promote the nutrients’ transportation. (ii) The mechanical properties of CNTs and their derivatives required further improvement i.e. up to the natural bone level in order to lower the conflict at the inorganic-organic interface. (iii) The early stages of cell culture in vitro is the center for most research investigations, hence detailed in vivo investigations should be focused on, followed by the polymeric materials’ degradation along with the discharge of CNTs.

304  Functionalized CNT for Biomedical Applications (iv) The long-term biocompatibility of CNTs based nanomaterials need to be addressed cautiously. Prior to any clinical trials, the entire knowledge of the biocompatibility of these nanomaterials should be focused on. Moreover, more efficient approaches to reducing their cytotoxicity need to be addressed. So that adverse effects of these materials could be minimized according to the precautionary principle during the fabrication, examining, and clinical applications. There is still a need to take a big step in the stability, utilization, and biosafety of these nanomaterials.

12.8 Conclusion In this chapter, we summarized the basic characteristics, structural, physical, and chemical properties, bone compatibility along with interactions and biodegradation of different types of CNTs based nanomaterials, and their typical applications in tissue engineering. Carbon nanotubes with excellent physiochemical characteristics such as electrical, thermal, mechanical, and structural properties have been extensively employed in electronic, photovoltaics, and energy storage applications and nowadays for the application of biomedical fields. Recently, CNTs are considered the most favorable candidates with immense potential for the successful engineering of fabricating to stimulate the adhesion, differentiation, and proliferation of injured tissue. CNTs based scaffolds permit the enhancement of the electrical activity as well as the direction of the nanotubes promoting tissue differentiation and growth. However, the main concern when employing pristine CNT constructs is the matter of toxicity. Numerous studies report that to minimize the toxicity and enhance the dispersity in the aqueous phase, CNTs can be modified with different functional groups (for instance -COOH and -OH and) using different approaches. Furthermore, the combination of CNTs along with the polymeric scaffolds for the fabrication of constructs for tissue engineering has been verified to improve viability, enhance mechanical integrity, improve electrophysiological performance along with better cell adhesion, and uniformity. For tissue engineering applications, CNTs have been primarily employed as reinforcement materials in the polymeric framework, revamping electrical and mechanical features and permitting the development of scaffolds for cardiac, bone, cartilage, and neural tissue engineering. The composition of polymer matrix with CNTs can enhance the nerve cell responses by accelerating the conductivity of the scaffold related to the nerve. It can also

CNT Applications in Tissue Engineering  305 promote the biological responses, elastic strength, and conductivity of the scaffolds related to the cardiac tissue. Additionally, CNTs are employed to reinforce the biological and mechanical features for bone tissue engineering. In the future, the development of nanotechnologies that are rationally designed and related to the current advancement in CNTs based nanostructures will solve most of the problems encountered in current tissue engineering applications.

Important Websites https://www.prescouter.com/2017/03/applications-carbon-nanotubes/ https://nanoten.com/NTSite/ https://www.nanowerk.com/nanotechnology/introduction/introduction_ to_nanotechnology_22.php https://www.nanocyl.com/expertise-center/health-safety-environment/

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13 Functionalized Carbon Nanotubes for Cell Tracking Sagar Salave, Dhwani Rana, Jyotsna Vitore and Aakanchha Jain* National Institute of Pharmaceutical Education and Research (NIPER)Ahmedabad, Palaj, Opp Air Force Station, Gandhinagar, Gujarat, India

Abstract

Carbon nanotubes owing to their attributes of high surface area, unique needle-like structure, and capability of circumventing the physical and biochemical blockage of the blood-brain barrier are extensively explored for theragnostic applications. Combining the surface of carbon nanotubes with desired functional groups imparts unique properties to carbon nanotubes and enhances their applicability. Functionalized carbon nanotubes of anticipated strength and conductivity properties provide us with options to explore. Functionalized carbon nanotubes-based biosensors are extensively used for diagnostic purposes and one wide application of functionalized carbon nanotubes is in cell tracking. We intend to comprehensively explore the application of functionalized carbon nanotubes for cell tracking and advances made in this regard. Keywords:  Carbon nanotubes, cell tracking, functionalization, surface coating, cell-specific tracking

Abbreviations CLSM Confocal laser scanning microscopy CNTs Carbon nanotubes FITC Fluorescein isothiocyanate GNT Gadonanotube HSPC Hematopoietic stem/progenitor cells LDH Layered double hydroxide MRI Magnetic resonance imaging PLPEG Phospholipid-polyethylene glycol *Corresponding author: [email protected]; [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (319–338) © 2023 Scrivener Publishing LLC

319

320  Functionalized CNT for Biomedical Applications SPIONs  Super magnetic iron oxide nanoparticles SW Single-walled

13.1 Introduction In all living organisms, cells are the structural and fundamental unit. Governing the morpho-dynamic behavior of cells remains the most crucial part of understanding the physiological processes and underlying cellular behavior in healthy as well as diseased conditions [1]. This presiding is at times carried out by imaging and analysis, typically involving tracking as well as quantification of multiple cells in phase-contrast microscopy or differential interference contrast microscopy, intravital microscopy, timelapse fluorescence, and computational algorithms [2, 3]. Individual cells vary substantially in their behavior and temporal evolution. These typical attributes help remarkably in not only identification but also understanding cell to cell differences and the prevailing heterogeneity amongst them [4]. Thus, cell tracking usually refers to the spatial-temporal studies of living cells’ motility [5]. These two aspects are the chief image processing stages in cell tracking. Cell segmentation, the spatial aspect, refers to the method of dividing the image into biologically significant segments and generating a new image whereas cell association, the temporal aspect, implies to the method of identification and linkage of segmented cells for obtaining cell trajectories from frame to frame in the image sequence. Briefly, segmenting individual cells and connecting cells over time is the usual cell Spatial-temporal studies of living cells motility

Cell tracking

Cell segmentation: Spatial aspect

Cell association: Temporal aspect

IMAGE ANALYSIS

Cellular information transmission through dynamic biochemical signaling networks Signaling Dynamics

Image cleaning

Background subtraction and drift correction

Identification of individual cell Image in every frame segmentation Capturing of whole cell or cellular compartments

Linking

Linking together all the detections for an individual cell Formation of temporally cohesive record for each cell

Quantitative information: Metrics of cellular morphology and fluorescnce intensity - over time Lineage information: Using suitable algorithm and data structure

Figure 13.1  Cell tracking basics.

Records

Functionalized CNTs for Cell Tracking  321 tracking in practice. Newer trends are evolving for improving robustness by employing the use of models and probabilistic estimation methods [2]. The collected dynamic data can be integrated with end-point genomic assays to discover and get a better understanding of cellular behavior [4]. Figure 13.1 represents the generalized concept of cell tracking and associated features. The usefulness of cell tracking lies in accurate capturing of the cellular events and their dynamics, cell behaviors, cellular motions, and quantification of migration, morphological changes, and rebuilding of cell lineages [1, 6, 7]. Some therapies like adoptive immunotherapy as well as cell-based therapy have been evaluated as new therapeutics in several clinical practices and diseases [5, 8–10]. Various cell-based therapeutic strategies require the determination of location, distribution, and viability by cells with their biological fortune relating to cellular activation as well as differentiation. Cell tracking methods serve to elucidate the new biological mechanism [11, 12]. There are two foremost doctrines of cell tracking [13]; i) Direct labelling and ii) Indirect labelling. Direct labelling is simple which does not require the genetic modification of the cell [14]. But the limitation of direct labelling system is the tendency of label to get diluted upon cell division resulting into a reduced number of labels in the individual cell. Additionally, the label gets disseminated asymmetrically to the progeny cells all through the cell division. Indirect labelling method uses introduction of a reporter gene into the cell followed by its consequent translation into enzymes, receptors, bioluminescent or fluorescent protein [15]. Based on the stability of the reporter gene, labelled cells could be observed throughout the complete life span of cells. Upon passing of this reporter gene onto the progeny cells, expanding cell population imaging is possible which gives the cell viability information.

13.2 Carbon Nanotubes Carbon nanotubes (CNTs) have long been referred to as versatile system due to their remarkable features in the domains of biological science, targeted drug delivery, and tissue engineering [16–18]. Figure 13.2 depicts the role of CNTs as a versatile platform for cell tracking. To date, a large variety of applications of CNTs has been explored to explain the mechanism of CNTs in various research areas [19]. Cell tracking stands as the most significant approach of CNTs for measuring various metabolic activity and live status of cells. CNTs pave the way for non-invasive cell tracking in the recent era [20]. It owns the characteristics features including high surface

322  Functionalized CNT for Biomedical Applications Bi3+ for X-ray CT Radionuclides for PET/SPECT

Iron oxide nanoparticles for MRI

Figure 13.2  Schematic representation of CNTs as a versatile platform for cell tracking.

area, unique – needle-like structure, ability to carry targeted moiety through the cell membrane. Single-walled (SW) and multiple walled CNTs are extensively explored as a biosensor in the medical field [21]. In current time, CNT-based bioimaging is widely investigated. SWCNTs exhibiting quasi-one dimensions possess a narrow band almost the 1eV that allows emission of fluorescence through NIR I region ranging from 700-900 nm whilst NIR-II region ranging from 1100-1400 nm. Due to the stronger absorbance at the NIR region CNTs play a crucial role as the photoacoustic imaging contrast agent. In addition, it is used in cell labelling grounded on Raman imaging, and magnetic resonance imaging (MRI) [22]. CNTs are known for their potential to uniquely equip and transfer the drug molecules across physiological membranes. Functionalized nanotubes, resulting from post-synthesis surface modification of nanotubes, are excellent carriers, efficient delivery systems, and have theragnostics applications [23]. Apart from these applications, CNT hybrids are explored for tracking administered nanocarriers for assessing in real-time their spatial distribution as well as hence measuring their biodistribution profile [24]. For the potential application of CNTs essential and wide functionalization to make them capable to tune their properties. Several strategies have been encountered to functionalize the CNTs as represented in Figure 13.3. These strategies are followed under two main categories [25]; i) Mechanical technique, and, ii) Chemical technique. The mechanical technique involves high shear missing, high impact mixing, ultracentrifugation, and sonication whereas the chemical method involves alteration of the surface energy of CNTs. Chemical modification aims for surface chemistry manipulations of CNTs by the method of adsorption

Functionalized CNTs for Cell Tracking  323 Covalent functionalization

Non-Covalent functionalization

Endohedral functionalization

External decoration

Figure 13.3  Commonly used strategies for CNTs functionalization.

involving non-covalently binding and covalent method involving functionalization of CNTs by covalent bonding [26]. Functionalized forms of CNTs may have different electrical, mechanical, and optical characteristics compared to the original form [27]. Lamm et al. studied the merits of fluorescence microscopy, as a method having powerful non-invasive way of penetrating nanoparticles in the physiological domains. They investigated induction of fluorescence energy transfer system amongst a molecule of lysophospholipid molecule and a SWCNT upon cellular uptake. They also described that coating nanotube with RNA and suspending fullerenes with phenolic acids facilitates their translocation through cellular membranes and shuttling amongst cellular organelles. Further, employing the use of molecular simulations could facilitate and guide such experimentations [28]. Moreover, owing to the exceptional attributes of CNTs, it has opened new avenues in the treatment of antimicrobial disorders [29, 30]. Jin-WooKim demonstrated the anti-microbial effects of CNTs shielded with bacterial filters. It was observed that bacteria bind the CNTs, as they possess high binding affinity and further get physically destroyed. The results of this study revealed that CNTs have the superior capability of NIR-responsive bunching PT contrast agents, that could be applied for the diagnosis of pathogenic organisms as well as antimicrobial agents at a single bacterium level [31]. Cellular mechanisms including cell internalization and uptake can be understood from the CNTs based cell tracking [24]. Numerous researchers have demonstrated the mechanism of cell permeability via cell membrane through passive transport or endocytosis. In addition, a reduction in cellular uptake has been observed with the help of fluorescently labelled macromolecules decorated on CNTs [32, 33].

324  Functionalized CNT for Biomedical Applications There are reports in the literature where two new CNT-based contrast agents were firstly synthesized by Wilson and co-workers and evaluated their performance in 2005. This material is used as a magnetic contrast agent, which is referred to as Gadonanotube (GNT). It has the length between 20-80 nm. GNTs exhibited the greatest T1relaxivity (r1) known, with values of up to 170 mM-1per s-1 per Gd3+ ion at 40 °C and 1.5 T. GNTs are amphiphilic in nature when disperse in surfactants which induced biocompatibility and hence it easily penetrated through local membrane. The synthesized GNTS consisting Gd3+-ion clusters or Gd3+ was further evaluated on mesenchymal stem cells MSCs. The results suggested that CNTs based contrast agent was taken up by mesenchymal stem cells (MSCs) in absence of any transfection agent and toxicity was not found on the either of used CNTs addition to this, Gd3+n-ion clusters containing ultra-short SWCNTs in the US-tubes are linear Superparamagnetic molecular magnets with MRI efficacies of 40 to 90 times larger than any Gd3+ based contrast agent in existing medical custom. Studies show that GNTs may denote a substantial revolution in design of contrast agent for high-field imaging [34]. Various ongoing research suggests the use of ferromagnetic metals such as Fe, Ni, Co for the synthesis of super magnetic iron oxide nanoparticles (SPIONs) straight in the apertures of silica nanotubes (SNTs). These silica nanoparticles have characteristic features of having good magnetic property. The integration also showed promising results in improvising the MRI contrast agent. In another work, Bai et al. [35] developed SPIONs straightway in the apertures of SNTs. In another report, amino group functionalized CNTs (f-CNTs) were detected in two different cell types i) multidrug-resistant cells (K562A) and, ii) the parent cells (K562S) at a different incubation temperature viz. 4°C rather than 37°C. This result suggests that fluorescent-tagged CNTs can aid the permeation through the cell membrane by transverse endocytosis [36]. Similarly, Kostarelos et al. elucidated the capability of fluorescently labelled CNTs to interact with cells and govern the important variables involved in these interactions. For this, they developed several f-CNTs such as ammonium-functionalized CNT, fluorescein isothiocyanate (FITC) f-CNT, CNT bi-functionalized with ammonium groups and FITC, acetamid of-CNT, CNT bi-functionalized with methotrexate, and FITC, shortened CNT bi-functionalized with amphotericin Band FITC, and shortened CNT bi-functionalized with ammonium groups and FITC using an amide linkage. Confocal laser scanning microscopy (Hematopoietic stem/progenitor cells) epifluorescence microscopy was employed to track and image the fluorescently labelled CNTs and/or inherently luminescent f-CNTs. Interactions amongst fluorescently labelled CNTs and an extensive

Functionalized CNTs for Cell Tracking  325 diversity of viable cells were then examined and it was found out that cellular internalization of fluorescently labelled CNTs occurs in the former ones. It was settled that fluorescently labelled CNTs hold a capability to be engulfed by prokaryotic as well as mammalian cells and to intracellularly traffic between various cell-based barriers through energy-independent processes [37].

13.2.1 Cellular Interaction of CNTs Cell trafficking of CNTs has immense implications in medical science and the crux of it lies in the communication of CNTs and cell membranes, controlled by the regulations of molecular cellular biology as well as the fundamental attributes of the nanotubes [11, 12, 38]. CNTs are endowed with super magnetic properties by the coating of magnetic nanoparticles on the surface [39, 40]. Owning to their magnetic properties, they are mostly having high reverse relaxivity [41]. Giuseppe Lamanna appraised the role of nanoparticle- CNT (NP/CNT) hybrids functioning like contrast agent for MRI along with their connections with cells. This multimodal complex opened the doors for new platform strategies in drug delivery as well as in theragnostic application. Although, there are various methods used for the fabrication of CNTs with NP resulting into extremely anisotropic ferromagnetic nanostructures. Ligand exchange or chemo-selective ligation method is preferred to use. The study results suggested that with this technique, well-organized, stable, and effective linking of magnetic iron oxide NPs to MWCNTs were obtained. The fabricated hybrid showed efficient internalization in tumor cells without indicating toxicity effects. It was observed that NP/CNTs hybrid could label cells and show magnetic mobility even at the single-cell level with high resolution of MRI. Biodistribution analysis also highlights the application of this tool in cell tracking [36].

13.3 Cellular Tracking via CNT Carbon nanotube having the size in the range of (~100 to 300 nm) has successfully been used for tracking the molecular motion of the living cells over time from millisecond to hours. In order to follow motorized movement in cells by fluorescence microscopy needs to fulfil certain criteria including (i) stable, non-bleaching fluorescent probes, (ii) effective targeting of probes to selective compounds, and (iii) high signal-to-noise ratio (S/N) in imaging. CNT-based tracking is invasive in nature and

326  Functionalized CNT for Biomedical Applications has been used in the observation of kinesin-1 motor protein in COS -7 cells. Cells are dynamic organizations and molecular force generation is a dynamic process at a supramolecular scale. CNTs can observe the directed ­kinesin-driven transport on microtubules in addition to that it can also observe the fluctuations due to moving kinesin bound to the microtubules. The microtubules track is embedded in the viscoelastic actin cytoskeleton, which turns fluctuation and generates stress by cytoplasmic myosin [42]. Gadolinium f-MWCNTs were developed to work like contrast agent ­intending MRI labelling as well as tracking [38]. Diethylenetriaminepentaacetic acid (DTPA) aiding tight attachment of gadolinium atom on the surface of nanotube was done. Figure 13.4 depicts development of DTPA modified MWCT for development of contrast agent. Developed contrast agents were known to be constant for about two weeks in water as well as mouse serum. Three-fold higher r1 relaxivity was observed compared to a clinically approved contract agent. The contrast efficiency investigated at 7T was obtained 6.6 mM-1s-1 in human umbilical vein endothelial cells [38]. In one learning, Singh et al. established the biopharmaceutical parameters of f-CNTs following i.v. administration. They functionalized water-­ soluble, SWCNT with a chelating molecule diethylenetriaminepentaacetic and labelled with indium7 (111In) for the purpose of imaging. The developed f-SWCNT was administered intravenously and was radioactively traced employing the use of gamma scintigraphy. This ensured that f-SWCNT was not retained in liver or spleen; organs related to reticuloendothelial system and were quickly cleared from the blood stream via the route of renal excretion. Also, urine excretion studies exploiting both f-SWCNT and f-MWCNT and electron microscopy analysis of the same discovered that both the nanotubes were expelled as intact nanotubes. This quick blood clearance along with a half-life of 3 hours indicated the clinical potential of CNTs [43]. Further, Wang et al. fabricated radio-labelled iron oxide COOH

Carboxylic acid modif ied MWNT

1

DTPA

NH3+

Amino group modif ied MWNT

2

Diethylenetriamine Penta acetic acid (DTPA) modif ied MWNT 3

Figure 13.4  Schematic representation of DTPA modified MWNT for MRI cell labelling and tracking.

Functionalized CNTs for Cell Tracking  327 decorated MWCNT as dual magnetic resonance and s­ ingle-photon emission computed tomography imaging materials.  Using in situ  generation hybrids of different amounts of Fe2O3 were synthesized and it was known from physicochemical characterizations that magnetic properties to hybrids were imparted due to the occurrence of SPION. These hybrids were then radio-labelled with technetium-99m via functionalized bisphosphonate and the quantitative biodistribution in mice was carried out using SPECT/CT imaging and γ-scintigraphy. In this examination, the existence of SPION was recognized by Perls stain whereas the presence of MWCNT was recognized using Neutral Red stain. Further, the histological examination revealed no abnormalities in mice. Transmission electron microscope images of tissues of spleen and liver discovered co-localization of SPION and MWCNT within the same intracellular vesicles, and hence indicated the in vivo solidity of the hybrids following intravenous injection. This study divulged the potential of SPION-MWCNT hybrids as dual MRI and SPECT contrast agents for in vivo use [44]. Lamanna et al. assessed the potential of SPION coated MWCNTs (NP/ CNT) hybrids as a contrast agent for MRI and interactions of the same with cells. Nanotubes covered with NP allowed conversing magnetic properties to CNTs. The capability of the hybrids to magnetically govern and influence cells was also inspected. It was found that NP/CNTs could be deployed using a remote magnetic field with improved contrast in MRI. The developed system showed internalization in the neoplastic cells without demonstrating significant cytotoxicity. The labelled cells displayed magnetic mobility and hence could be magnetically manipulated perceived at a single cell level through high-resolution MRI [36]. T.S.T. Balamurugan et al. prepared the Prussian blue microcubes functionalized graphenated nanotubes modified electrodes to track the in vivo H2O2 generation in mammalian cells. The synthesis process was straightforward and the fabricated sensor presented detection limit at nanomolar range, reproducibility, and durability [45]. Besides cell tracking, CNTs possess potential applications in therapeutic modalities. Emma Mooney et al. have established a study demonstrating the effect of CNT on hMSC renewal, cellular ultra-structure, nuclear location metabolic activity, and differentiation. Screening based on conductance value among the various CNTs such as SWCNT, MWCNT, COOHf-SWCNT, and OH f-MWCNT was done to recognize the optimal kind of CNT for usage with hMSC. It has been stated that COOHfSWCNT showed minimum toxicity to the cells. In this research, the f-CNT was distributed in hMSC media for the evaluation of all parameters. COOH functionalized and OHf-SWCNT showed a harmful outcome on

328  Functionalized CNT for Biomedical Applications the cells. However, at low concentration (0.00128 mg/mL) viability of cells and metabolic rate was enhanced compared with control. At high concentration (0.032 mg/mL) both OH functionalized and COOHf-CNTs found detrimental effect. The cells were microscopically analyzed and COOHf-WCNT was biotinylated with 1 × 103 mol NHS biotin for analysis. The study revealed that SWCNT travelled through the cell wall to a nucleus 24 hours later. Authors have explained that an innovative electrophysiological milieu for electrically stimulating HMSC encourage differentiation towards a cardiomyocyte linkage [46].

13.3.1 Effect of the Surface Coating of CNTs in Single-Particle Tracking Gao et al. evaluated different SWCNT external modifications for ­single-particle tracking applications in complex biomedical samples. The study involved screening various coatings in order to encapsulate the CNTs having luminescent property and displaying nominal cell related toxicity and negligible non-specific interactions with the cell membrane. Amongst all the screened coatings, phospholipid-polyethylene glycol (PLPEG) and pluronic F108-coated SWCNTs exhibited minimal acute cellular cytotoxicity of 1–4 days at 1 μg/mL and least cell-based interaction when compared to some other extensively utilized biodegradable surface coatings. Also, PLPEG-coated SWCNTs displayed brighter luminescence than F108-coated CNTs and could be viewed at video rate for some time at the single tube level while diffusing in a composite aqueous network and hence concluded to be the optimum coating for single nanotube tracking application in biomedical applications [47].

13.4 3D Tracking Using CNTs Reuel et al. established 3D tracking of fluorescent SWCNT following the use of an orbital tracking microscope within live HeLa cells. The tracking system had engrained spatial resolutions of 7-15 nm and a temporal resolution of 32 ms for visualization of chitosan-wrapped SWCNTs. The study involved the determination of the viscosity regime (above 250 cP) and the rotational diffusion coefficient could be used for estimation of length. The obtained tracking data were used for spatially mapping of corral volumes, determining active transport velocity, and calculating local viscosities within the cell. Within the cell, the coral volume was found to

Functionalized CNTs for Cell Tracking  329 be 0.27- 1.32 μm3, active transport velocity was found to be 455 nm/s and local viscosity was 54-179 cP. The SWCNTs could also be used as sensors in living cells, changing the fluorescence signal by about 4-13% in order to permit disjunction of the sensor signal from fluctuations owing to rotation of the SWCNT while gauging with a time resolution of 32 ms [22].

13.4.1 Detection of Single Protein Molecules Through CNTs Imaging and tracking of single-molecule coupled with florescent probes are known as single-molecule tracking. SMT eases the investigators to straightway witness molecular behavior and interaction in the living cells. Several molecular interactions and reactions occur in the living cells. SMT allows the researcher to observe distinct molecules at work in living cells precisely [48]. In 2017, MIT chemical engineers established arrays of CNT sensors that could detect single protein molecules being secreted from cells. These chemically modified CNTs could be consumed for protein production by distinct cells. This kind of fabrication can be employed for tracking even minor quantities of protein like tracking viral infection, nursing cells’ production of effective proteins, or even learning contamination of edible stuffs. Upon functionalization of these sensitive molecular sensing platforms-­ CNTs, one could observe the stochastic fluctuations of single molecules attaching to them [49]. In this regard, Landry et al. showed label-free recognition of specific proteins from bacteria-E. coli and yeast-P. pastoris immobilized in a microfluidic chamber, evaluating efflux of protein from individual organism in real-time. The array was invented leveraging the use of non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive SWCNT, employing an adjustable chemical spacer that could optimize sensor response. From various cell lines, proteins like unlabeled RAP1 GTPase and HIV integrase were specifically distinguished using large near-infrared fluorescent turn-on responses [50]. Moreover, the current research area also focuses the H2s monitoring in the biomedical domains due to the generation of a third endogenous gasotransmitter. Muhammad Asif et al. have demonstrated a simple strategy with the help of a sensitive electrochemical sensor and 2Dnanosheet-shaped layered double hydroxide (LDH) covered with CNTs nanohybrid (CNTs@ LDH). The synthesis was carried out by the coprecipitation method. The results showed that NTs@CuMn-LDH nanohybrid possess noteworthy sensing attributes for H2S determination. The lower detection limit was 0.3 nM (S/N = 3), with greater specificity, durability, and reproducibility.

330  Functionalized CNT for Biomedical Applications This structural integration strategy was a new platform to detect the abiotic H2s efflux formed by sulfate-reducing bacteria upon use in real-time assay [51].

13.4.2 Stem Cell Labeling and Tracking Through CNTs Uludag et al. presented an extremely effective and harmless mCNT-­ mediated stem cells labelling techniques. Fluorescent and magnetic FITCmCNTs were synthesized and magnetic-field mediated hematopoietic stem/progenitor cells (HSPC) uptake of FITC-mCNT was observed. The developed system was sent for fluorescence-activated cell sorter analysis for determining the uptake efficiencies. The results were suggestive of efficient internalization of FITC-mCNT by HSPC in a time and concentration-dependent manner. Compared to various nanoparticular uptake using innate or modified SPION, FITC-mCNT labelling or uptake was found more effective for the purpose of labelling stem cells. Moreover, the developed CNT showed no adverse effect on macrophages, no noteworthy outcome on chondrogenic, osteogenic, or adipogenic differentiation of hMSC, and was found non-cytotoxic [52]. Vittorio et al. inspected the role of MWCNTs with low metal impurities (2.57% iron) as MRI contrast agents for tracking stem cells. Cellular viability and proliferation was not seen to be affected by the developed MWCNTs, suggesting that such attributed exhibited by MWCNTs could aid in in vivo purpose of stem cell tracking/imaging as well as in MWCNTmediated targeted electro-chemotherapeutics [53].

13.4.3 Labelling and Tracking of Human Pancreatic Cells Through CNTs In one study, Syed et al. compared the SPION ferumoxide (Endorem) and MWCNTs for islet cell labeling as well as tracking. They incubated INS-1 E cells and human pancreatic islets obtained from twelve non-diabetic cadaveric organ donors with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days for assessing cell survival, function, beta-cell morphology, ultrastructure, and in-vitro and in-vivo MRI. Well-maintained morphology, as well as ultrastructure, was observed for both INS-1 E and human islets at the time of incubation in light and electron microscopy examination. The presence of MWCNTs was observed within beta cells whereas alpha cells were devoid of it when seen under electron microsocpe. Effective transplantation of MWCNT labelled human islets into subcutis of rats localized in the anticipated site was carried out employing the use of magnetic field while tracking was performed using MRI. The findings were

Functionalized CNTs for Cell Tracking  331 suggestive that MWCNTs could be a potential labelling multifarious and could be used along with human islets for experimental as well as transplantation studies [54]. This study highlighted the possible mechanism of CNT for cell internalization. The very first-time MWCNTs were reported for entering human beta cells through direct membrane penetration and endosomal pathways. This study highlights the potential of MWCNTs for islets grafting tools in tracing after engraftment and post-transplant monitoring.

13.4.4 CNT as Macrophage Carrying Microdevices Cell tracking techniques employing the use of MRI and positron emission tomography require ionizing radiation which is not the case with Raman imaging. In fact, Raman imaging is referred to be extremely material-­dependent, could be excited by NIR permitting deep tissue penetration at the centimeter scale. CNTs on the other hand display strong ­resonance-enhanced Raman modes possessing an exclusive Raman spectrum. Wang et al. developed an adaptable system to fabricate the microdevices and prepare microdevices-live cells complexes. This technique was further extended for the production of CNT built microdevices for tracking single macrophages using Raman scattering. The fabricated microdevices consisted of polyelectrolyte trilayer, CNTs, and PLGA. The generated Raman signals by the developed CNTs system in microdevices were perceived at a centimeter scale with the sample-lens distance under NIR excitation, suggesting its in-vivo potential. The fabricated microdevices were taken up by the principal macrophages while not affecting the cell viability as well as phagocytic capability of the cells. These single macrophages having microdevices could be spotted by Raman scattering and hold potential for non-invasive multiplex tracking of single macrophages in vivo [55].

13.4.4.1 Intracellular Fluctuations and CNT A quantifiable investigation focusing on molecular motions in cells was carried out by Fakhri et al., wherein the authors employed extremely steady near-infrared luminescence of SWCNTs targeted to kinesin-1 motor proteins in COS-7 cells. The authors stated that scheme of active random “stirring” institutes a mean of transportation, which differ from thermal diffusion as well as directed motor activities. The observed high-frequency action was thermally driven. Non-equilibrium dynamics were observed higher than 100 milliseconds. Apart from this author also observed robust

332  Functionalized CNT for Biomedical Applications random dynamics obliged by myosin that lead to increased nonspecific transport [56].

13.4.5 Limitations of CNTs CNTs are explored versions of carbon allotropes found in the same category of fullerenes. Various MWCNTs are regarded to cause cell-cycle arrest, perturbations in multiple cellular pathways, increase in ­apoptosis/ necrosis, and stimulation of genes concerned in cell-cycle regulation, cellular metabolism, transport, and reaction to stress. There are reports regarding SWCNT for topical skin application [57]. However, exposure to this material on the skin surface can lead to skin-associated issues including carbon fiber dermatitis, hyperkeratosis, etc. There are diverse studies reported with toxicity evaluation of CNTs. For instance, SWCNT has been studied on cell culture of immortalized human epidermal keratinocytes. It revealed that on the exposure up to 18 hours on cell culture, respective parameters such as oxidative stress as well as cellular toxicity were observed. Despite these, multiple reports are found in the literature for the mixture of Ferric compound with CNTs causing iron associated overload cell damage and provoking the other clinical condition such as heart diseases, cancers, hormonal abnormalities, skin diseases, immune system dysfunctions, liver diseases and diabetes [58]. The iron catalyst compounds inside the SWCNT material are sheathed in a carbon-based material. However, the nano-sized particles have been reported more toxic because of their large surface area.

13.5 Concluding Remarks and Future Perspective Functionalization and designating of CNTs have been proved as versatile and highly efficient strategy for enhancing the CNT application in the biomedical field. Surface modification is novel emerging approach to improve the theragnostic potential of CNTs as it influences biocompatibility, targeting and organ distribution. The applicability of f-CNTs in the cell tracking is gaining researchers’ interest. However, cell tracking is the sophisticated and invasive tool to monitor the particles and cellular components at cellular and molecular level. Novel approaches have been listed in this chapter with the desired fabrication specification of CNTs in terms of cell tracking application. Numerous microscopic and fluorescent techniques are used in reported literature to continuously monitor the cells internal environment and cellular uptake mechanism. The cell specific application of f-CNTs is

Functionalized CNTs for Cell Tracking  333 illustrated with their examples and results. Many researchers have claimed positive results for the cell viability and internalization of CNTs. Besides that, it is important to consider the cellular toxicity that can causes due to the various functional groups and carbon containing skeleton of CNTs. In addition, due to its nanoscale size range, after internalization into the cells, the CNTs interact with the living organism and might cause toxicity. Hence, the understanding and exploring the toxicity events and its exact mechanism followed by administration helps to fabricate and select the safe and efficient materials in the development of CNTs. In future, there is scope for exploration of different functionalized material for the cell tracking application by invasive way.

Important Links https://dspace.mit.edu/handle/1721.1/73371 ht t p s : / / s c h o l a r s h ip. r i c e . e du / bit s t re a m / h a n d l e / 1 9 1 1 / 9 0 9 4 0 / RevisedBIOMATERIALS.pdf?sequence=4&isAllowed=y https://tigerprints.clemson.edu/cgi/viewcontent.cgi?referer=https://www. google.com/&httpsredir=1&article=1279&context=all_dissertations

Acknowledgment The authors are thankful to former Director, NIPER-A for providing necessary literature search facilities.

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334  Functionalized CNT for Biomedical Applications An objective comparison of cell tracking algorithms. Nat. Methods, 14, 12, 1141, 2017. 4. Moen, E., Borba, E., Miller, G., Schwartz, M., Bannon, D., Koe, N., Camplisson, I., Kyme, D., Pavelchek, C., Price, T., Kudo, T., Pao, E., Graf, W., Van Valen, D., Accurate cell tracking and lineage construction in live-cell imaging experiments with deep learning. bioRxiv, 803205, 2019. 5. Svensson, C.M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M.T., Untangling cell tracks: Quantifying cell migration by time lapse image data analysis. Cytom. Part A, 93, 3, 357–370, 2018. 6. Majumdar, R., Steen, K., Coulombe, P.A., Parent, C.A., Non-canonical processes that shape the cell migration landscape. Curr. Opin. Cell Biol., 57, 123– 124, 2019. 7. Shrier, D., Tompsett, M., Shrier, L., Adult mother–daughter relationships: A review of the theoretical and research literature. Guilford Press. J. Am. Acad. Psychoanal. Dyn. Psychiatry, 32, 1, 91–115, 2004. 8. Cheng, M., Chen, Y., Xiao, W., Sun, R., Tian, Z., NK cell-based immunotherapy for malignant diseases. Cell. Mol. Immunol., 10, 3, 230, 2013. 9. Zhao, L. and Cao, Y.J., Engineered T cell therapy for cancer in the clinic. Front. Immunol., 10, 2250, 2019. 10. Xie, G., Dong, H., Liang, Y., Ham, J.D., Rizwan, R., Chen, J., CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine, 59, 102975, 2020. 11. Kircher, M.F., Gambhir, S.S., Grimm, J., Noninvasive cell-tracking methods. Nat. Rev. Clin. Oncol., 8, 11, 677–688, 2011. 12. Srinivas, M., Boehm-Sturm, P., Figdor, C.G., de Vries, I.J., Hoehn, M., Labeling cells for in vivo tracking using 19F MRI. Biomaterials, 33, 34, 8830– 8840, 2012. 13. Hong, H., Yang, Y., Zhang, Y., Cai, W., Non-invasive cell tracking in cancer and cancer therapy. Curr. Top. Med. Chem., 10, 12, 1237, 2010. 14. Kim, M.H., Lee, Y.J., Kang, J.H., Stem cell monitoring with a direct or indirect labeling method. Nucl. Med. Mol. Imaging (2010), 50, 4, 275, 2016. 15. Weissleder, R., Moore, A., Mahmood, U., Bhorade, R., Benveniste, H., Chiocca, E.A., Basilion, J.P., In vivo magnetic resonance imaging of transgene expression. Nat. Med., 6, 3, 351–354, 2000. 16. Zare, H., Ahmadi, S., Ghasemi, A., Ghanbari, M., Rabiee, N., Bagherzadeh, M., Karimi, M., Webster, T.J., Hamblin, M.R., Mostafavi, E., Carbon nanotubes: Smart drug/gene delivery carriers. Int. J. Nanomedicine, 16, 1681, 2021. 17. Simon, J., Flahaut, E., Golzio, M., Overview of carbon nanotubes for biomedical applications. Mater. (Basel), 12, 4, 624, 2019. 18. Dubey, R., Dutta, D., Sarkar, A., Chattopadhyay, P., Functionalized carbon nanotubes: Synthesis, properties and applications in water purification, drug delivery, and material and biomedical sciences. Nanoscale Adv., 3, 20, 5722– 5744, 2021.

Functionalized CNTs for Cell Tracking  335 19. Zare, H., Ahmadi, S., Ghasemi, A., Ghanbari, M., Rabiee, N., Bagherzadeh, M., Karimi, M., Webster, T.J., Hamblin, M.R., Mostafavi, E., Carbon nanotubes: Smart drug/gene delivery carriers. Int. J. Nanomed., 16, 1681–1706, 2021. 20. Hong, H., Yang, Y., Zhang, Y., Cai, W., Non-invasive cell tracking in cancer and cancer therapy. Curr. Top. Med. Chem., 10, 12, 1237–1248, 2010. 21. Sireesha, M., Jagadeesh Babu, V., Kranthi Kiran, A.S., Ramakrishna, S., A review on carbon nanotubes in biosensor devices and their applications in medicine. Nanocomposites, 4, 2, 36–57, 2018. 22. Reuel, N.F., Dupont, A., Thouvenin, O., Lamb, D.C., Strano, M.S., Threedimensional tracking of carbon nanotubes within living cells. ACS Nano, 6, 6, 5420–5428, 2012. 23. Cui, H.F., Vashist, S.K., Al-Rubeaan, K., Luong, J.H.T., Sheu, F.S., Interfacing carbon nanotubes with living mammalian cells and cytotoxicity issues. Chem. Res. Toxicol., 23, 7, 1131–1147, 2010. 24. Costa, P.M., Bourgognon, M., Wang, J.T.W., Al-Jamal, K.T., Functionalised carbon nanotubes: From intracellular uptake and cell-related toxicity to systemic brain delivery. J. Control. Release, 241, 200–219, 2016. 25. Mallakpour, S. and Soltanian, S., Surface functionalization of carbon nanotubes: Fabrication and applications. RCS Adv., 8, 109916–109935, 2016. 26. Meng, L., Fu, C., Lu, Q., Advanced technology for functionalization of carbon nanotubes. Prog. Nat. Sci., 19, 7, 801–810, 2009. 27. Norizan, M.N., Moklis, M.H., Ngah Demon, S.Z., Halim, N.A., Samsuri, A., Mohamad, I.S., Knight, V.F., Abdullah, N., Carbon nanotubes: Functionalisation and their application in chemical sensors. RSC Adv., 10, 71, 43704–43732, 2020. 28. Lamm, M.H. and Ke, P.C., Cell trafficking of carbon nanotubes based on fluorescence detection. Methods Mol. Biol., 625, 135–151, 2010. 29. Seo, Y., Hwang, J., Kim, J., Jeong, Y., Hwang, M.P., Choi, J., Antibacterial activity and cytotoxicity of multi-walled carbon nanotubes decorated with silver nanoparticles. Int. J. Nanomed., 9, 1, 4621–4629, 2014. 30. Liu, D., Mao, Y., Ding, L., Carbon nanotubes as antimicrobial agents for water disinfection and pathogen control. J. Water Health, 16, 2, 171–180, 2018. 31. Kim, J.W., Shashkov, E.V., Galanzha, E.I., Kotagiri, N., Zharov, V.P., Photothermal antimicrobial nanotherapy and nanodiagnostics with self-­ assembling carbon nanotube clusters. Lasers Surg. Med., 39, 7, 622–634, 2007. 32. Kam, N.W.S. and Dai, H., Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc., 127, 16, 6021–6026, 2005. 33. Xiao, H., Yang, L., Zou, H., Yang, L., Le, X.C., Analysis of oxidized multiwalled carbon nanotubes in single K562 cells by capillary electrophoresis with laser-induced fluorescence. Anal. Bioanal. Chem., 387, 1, 119–126, 2006.

336  Functionalized CNT for Biomedical Applications 34. Sitharaman, B., Kissell, K.R., Hartman, K.B., Tran, L.A., Baikalov, A., Rusakova, I., Sun, Y., Khant, H.A., Ludtke, S.J., Chiu, W., Laus, S., Tóth, E., Helm, L., Merbach, A.E., Wilson, L.J., Superparamagnetic gadonanotubes are high-performance MRI contrast agents. Chem. Commun. (Camb), 32, 3915–7, 2005. 35. Issa, B., Obaidat, I.M., Albiss, B.A., Haik, Y., Magnetic nanoparticles: Surface effects and properties related to biomedicine applications. Int. J. Mol. Sci., 14, 14, 2013. 36. Lamanna, G., Garofalo, A., Popa, G., Wilhelm, C., Bégin-Colin, S., FelderFlesch, D., Bianco, A., Gazeau, F., Ménard-Moyon, C., Endowing carbon nanotubes with superparamagnetic properties: Applications for cell labeling, MRI cell tracking and magnetic manipulations. Nanoscale, 5, 10, 4412–4421, 2013. 37. Kostarelos, K., Lacerda, L., Pastorin, G., Wu, W., Wieckowski, S., Luangsivilay, J., Godefroy, S., Pantarotto, D., Briand, J.P., Muller, S., Prato, M., Bianco, A., Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nat. Nanotechnol., 2, 2, 108–113, 2007. 38. Servant, A., Jacobs, I., Bussy, C., Fabbro, C., Ros, T., Pach, E., Ballesteros, B., Prato, M., Nicolay, K., Kostarelos, K., Gadolinium-functionalised multiwalled carbon nanotubes as a T1 contrast agent for MRI cell labelling and tracking. Carbon, 97, 126–133, 2016. 39. Zhou, H. and Wang, X., Decoration of magnetic nanoparticles on functionalized multi-wall carbon nanotubes by in situ method. Adv. Mater. Res., 601, 37–40, 2013. 40. Masotti, A. and Caporali, A., Preparation of magnetic carbon nanotubes (Mag-CNTs) for biomedical and biotechnological applications. Int. J. Mol. Sci., 14, 12, 24619, 2013. 41. Tomczyk, M.M., Boncel, S., Herman, A., Krawczyk, T., Jakóbik-Kolon, A., Pawlyta, M., Krzywiecki, M., Chrobak, A., Minoshima, M., Sugihara, F., Kikuchi, K., Kuźnik, N., Oxygen functional groups on MWCNT surface as critical factor boosting T2 relaxation rate of water protons: Towards improved CNT-based contrast agents. Int. J. Nanomed., 15, 7433–7450, 2020. 42. Yokomizo, H., Possingham, H.P., Hulme, P.E., Grice, A.C., Buckley, Y.M., Olofson, P., Grijalva, E., Kerr, D., Hogle, I., Thornton, W., Fakhri, N., Wessel, A.D., Willms, C., Pasquali, M., Klopfenstein, D.R., MacKintosh, F.C., Schmidt, C.F., We thank the personnel of the invasive spartina project, including high-resolution mapping of intracellular fluctuations using carbon nanotubes. Biol. Invasions, 127, 839–849, 2006. 43. Singh, R., Pantarotto, D., Lacerda, L., Pastorin, G., Klumpp, C., Prato, M., Bianco, A., Kostarelos, K., Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proc. Natl. Acad. Sci., 103, 9, 3357–6662, 2006. 44. Wang, J.T.W., Cabana, L., Bourgognon, M., Kafa, H., Protti, A., Venner, K., Shah, A.M., Sosabowski, J.K., Mather, S.J., Roig, A., Ke, X., Van Tendeloo,

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338  Functionalized CNT for Biomedical Applications 57. Ding, L., Stilwell, J., Zhang, T., Elboudwarej, O., Jiang, H., Selegue, J.P., Cooke, P.A., Gray, J.W., Chen, F.F., Molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast. Nano Lett., 5, 12, 2448–2464, 2005. 58. Shvedova, A., Castranova, V., Kisin, E., Schwegler-Berry, D., Murray, A., Gandelsman, V., Baron, P., Exposure to carbon nanotube material: Assessment of nanotube cytotoxicity using human keratinocyte cells. J. Toxicol. Environ. Health A, 66, 20, 1909–1926, 2011, https://doi.org/10.1080/713853956.

14 Functionalized Carbon Nanotubes for Treatment of Various Diseases Ajahar Khan*, Khalid A. Alamry and Raed H. Althomali Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract

In present times functionalized carbon nanotubes (CNTs) have received more attention from therapeutic formulation scientists due to their unique intrinsic physical and chemical characteristics. The aspect ratio is notion to be accountable for their brilliant cell penetration ability, semi/anisotropic conductivity, as well as their axis, is best for integration with muscular and nervous tissue, and ability to penetrate biological membranes. These unique features of functionalized-CNTs enable them for a wide range of biomedical applications, such as infectious diseases, cancer remediation, diagnosis, delivery of drugs, and central nervous system disorders applications. Their adaptable physicochemical properties facilitate the introduction of various pharmaceutically significant. CNTs can be easily functionalized with different functional groups to allow customizing of biological recognition. Herein, we summarized widely the characteristics of CNTs, such as their mechanisms performance, and huge potential in biomedicine. This work also describes several functionalization approaches utilized for CNTs that originated new opportunities for researchers to enhance the potential of delivered therapeutics. Consequently, the previous reports have surveyed the current progress to emphasize the current application status in pharmaceuticals. The potential biomedical applications of CNTs with their biosafety/toxicology description and the way to conquer the cytotoxicity are also reviewed with future perspectives. Keywords:  Diagnosis, functionalized-CNTs, cancer treatment, gene delivery, biomedical applications

*Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (339–376) © 2023 Scrivener Publishing LLC

339

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14.1 Introduction The promising field of nanomaterials bridges the physical sciences with biological sciences using chemical techniques in emergent novel platforms and devices for disease diagnosis and remediation by understanding biological systems [1]. Nanomaterials comprise sizes in the range of 1 to 100 nanometers, similar to various biological macromolecules for instance deoxyribonucleic acid (DNA) plasmids, antibodies, and enzymes. Therefore nanomaterials demonstrate attractive physical characteristics, different from both the molecular and bulk scales, exhibiting innovative spaces for the research and application of the biomedical-related fields. There are numerous types of nanomaterials already studied or under research that could be considered for biomedical applications [2–4]. With the increasing demands of potential nanomaterials, carbon nanotubes (CNTs) are reviewed as the most promising candidate in the nanotechnology field and the construction of CNT-based materials is estimated to produce huge societal interests [5]. CNTs have concerned the interest of researchers worldwide; their incredible strength, wonderful physico-­chemical features, and miniature size make them particularly useful for different functions. Additionally, owing to unique surface properties like thermal, electrical and optical properties, CNTs have attained considerable interest as an imaging and diagnosis agent [6, 7] in the field of biomedical and became a most wonderful nanomaterial in recent drug development [8]. CNTs were accidentally discovered as a by-product of fullerene by Sumio Iijima in 1991 at the time of study surfaces of graphite electrodes during electric arc discharge [9]. This study along with the structural analysis of nanotube offered a novel course to the research of carbon, which harmonized the interest for and actions within organized research of fullerene. Based on different arrangements in graphene cylinders, two types of CNTs are found i.e., single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) classified based on the numbers of carbon layers present. A single, seamless and uniform graphene tube consisting of a single layer of carbon atoms is SWNTs and in comparison to SWNTs, MWNTs are formed by multiple, single concentric graphene tubes attaining several layers of carbon atoms and have much larger diameters [10]. While MWNTs attain less affluent and striking optical features than SWNTs, their performance in living environments can be diverse from that of SWNTs because of their bigger sizes, which can provide dissimilar substrates for different causes, including delivery of bigger biomolecules such as DNA plasmids into cells [11, 12]. CNTs’ solubility is a great challenge in executing several research formulations but covalent functionalization of

CNTs for Treatment of Various Diseases  341 CNTs can resolve the problem of solubility in a better way. Furthermore, functionalization with triethylene glycol, polyethylene glycol, and with proteins/oligonucleotides biomaterials could also enhance the solubility of CNTs [13]. A broad variety of bioactive materials can be conjugated with the CNTs and provide new routes in pharmaceutical applications. The construction of the bioelectronic device has allowed better chances concerning the immobilization of proteins and DNA over the CNTs surface. The CNTs also exhibit superb anti-oxidant activity against photo and thermal degradation of polymers [14]. The excellent electrical conductivity allows CNTs to be used as optical and electronic biosensors for the detection of microorganisms and bioactive molecules such as cells, DNA, and proteins. CNTs exhibit brilliant features of high optical absorption in the Raman scattering [15], photoluminescence [16], infrared region [17], echogenic, and photoacoustic features which are beneficial in tracking and visualization of nanotubes in the living system. In addition to the above-raised characteristics, the healthcare applications of CNTs in vaccine delivery, neural regeneration [18], gene and drug delivery, muscle and bone regeneration [19], and tissue engineering [20], are key achievements of researchers. CNTs can be utilized for the cause of diagnosis as a therapeutic imaging agent, biomolecular detector, nanotweezers, and biosensor [21]. The diminished size of particles of CNTs promotes the surface area contact then redoubles the uptake through the cellular membrane. Hydrophobic nature and size in nano range help CNTs in improved cellular internalization, manifesting nanotubes influence as significant carriers towards brain delivery [22]. Straightforward functionalized-CNTs produce them vital carriers for specific delivery of anticancer genes and drugs with lower adverse effects [23]. In biology and medicine, CNTs have been widely used as nanocarriers to deliver various proteins, peptides, small interfering ribonucleic acids (siRNAs), and plasmid DNAs [24]. Furthermore, hybridization and composition with many metallic nanoparticles, various biomaterials, and polymers produce them alter the properties of CNTs which improves its application in biology and medicine [25]. This chapter plans to briefly emphasize the current state of investigations and recent advances into the promising biological consequences of CNTs, with a look at how their biocompatibility is suitable for disease treatment. Furthermore, the significance of CNT composites their toxicities studies for in vivo and in vitro cellular uptake has been discussed. Sidewise an insight into how to enhance the suitability, comparability, reliability, and validity, of currently used examination techniques for establishing the possible biological effects of CNTs has also been reviewed.

342  Functionalized CNT for Biomedical Applications

14.2 CNTs: Basic Structure, and Synthesis Methods 14.2.1 Structure and Synthesis of CNTs As previously discussed, during fullerene synthesis CNTs were accidentally discovered as a by-product [9]. CNTs are graphene’s covered sheets comprising 2 end caps analogous to semi-C60 [9] and classified into two types i.e., SWNTs are single graphene cylinders, flexible, smaller in diameter, and allow imaging assist while MWNTs are a complex nesting of graphene cylinders with large surface area and, highly proficient endohedral filling [26, 27]. The CNTs possess a diameter in the range of 0.4-100 nm, while can attain a length up to diverse micrometers. In order to synthesize CNTs, three main methods i.e., laser-ablation, arc-discharge, and catalytic growth (chemical vapor deposition) are established and proposed over the last two decades. CNTs can be synthesized in 2 forms i.e. as a powder (substrate-­ free) and on the substrate. The substrate comprises vertically aligned CNTs, which are commonly used for electronic and sensors systems.

14.2.2 Arc Discharge Technique The first time the CNTs were synthesized through the arc discharge technique [9]. In the arc discharge technique carbon is vaporized in the inert atmosphere (argon or helium) from a graphite cathode heated through applied current. Herein, the function of higher pressure (50-700 mbar) and high-current-voltage (50-100 amps) creates highly-temperature discharge among two electrodes positioned 1 mm distant, which leads to evaporation of carbon rods followed by depositing on the rod-shaped cathode tubes. Several metal catalysts for example Pt, Co, Fe, Rh, or Ni, are used for SWCNTs synthesis. The properties of CNTs may be adjusted by introducing catalysts in the cathode and by changing the current intensity [26].

14.2.3 Laser Ablation Technique In this technique, a continuous laser of strong power is applied to vaporize targeted graphite within a furnace at a temperature of 1200°C with helium or argon gas at a pressure of 500 torr. The CNTs synthesized by the laser ablation technique exhibit high purity, controlled size with lesser structural defects, chirality, and around 70% yield [28]. The laser ablation technique can be employed for the synthesis of both SWNTs and MWNTs. By this method CNTs of fine crystallinity can be developed, though, fullerenes, amorphous carbon, and catalysts are left as the final product.

CNTs for Treatment of Various Diseases  343

14.2.4 Catalytic Chemical Vapor Deposition Technique This technique is focused on the catalytic decomposition of a gas comprising carbon on a heterogeneous or homogeneous catalyst. In catalytic chemical vapor deposition technique, the hydrocarbon molecules (such as acetylene, benzene, ethane, and toluene, etc.) are dissociated into the reactive carbon compound around 500–1000°C in a quartz tube positioned in the furnace. Nucleation of the catalyst through chemical etching by thermal annealing or ammonia is accomplished after the catalyst deposition on the substrate. The carbon resource persists in the gas phase in the reaction chamber and is converted to the atomic level, that diffuses for the metal catalyst on which the development of CNT arises [29]. The CNTs synthesized by catalytic chemical vapor deposition technique can be lengthening in millimeters range. The temperature required in this technique is lesser than that desired for the laser ablation and arc discharge techniques; however, these CNTs can display various limitations. In a report, Araga et al. studied the synthesis of MWNTs by using plasma-enhanced chemical vapor deposition method at relatively low temperature (450°C) straight on the charcoal surface derived from coconut-­shell [30]. Herein, minerals including potassium, sodium, magnesium, and calcium found in the biomass behave like catalysts, and the low temperature terminate the catalytic behavior of Fe. The MWNTs synthesized, which were characterized by Raman spectroscopy, X-ray spectroscopy, elemental detection, and scanning electron microscopy exhibited smaller diameters (25–30 nm) [30].

14.3 Functionalization of CNTs Even surfaces and inherent hydrophobicity with no hanging bonds present, pure CNTs are incompatible and chemically inert toward nearly all inorganic and organic solvents and exceptionally tricky to disperse in aqueous phases. These limitations have produced a major drawback for CNTs applications in biology and medicine. Therefore, it is needed to carry out further modification on the surface of CNTs that not only enhance their miscibility/dispersibility but also make them able to interact with biological systems [31]. Nowadays, scholars have already studied that in both types of CNTs carbon atoms reveal chemical reactivity for several reagents and therefore may be believed as newly hybrid types of macromolecular carbon form. After functionalizations, the biocompatibility and miscibility of CNTs in water might be improved. In this regard, two main

344  Functionalized CNT for Biomedical Applications approaches i.e. covalent and non-covalent functionalizations can be carried to improve their water solubility [32].

14.3.1 Covalent Functionalization Covalent functionalizations could be designated as ‘defect functionalizations’ as barely faulty C-atoms at the end or on the sidewall of CNTs might be oxidized using good oxidants to produce carboxylated fractions or carboxylic acid groups, which can be modified chemically through esterification or amidation [33]. The covalent modifications on the sidewall carbon atoms are accomplished by rehybridization or converting the configurations of sp2 carbon into sp3, hence, producing covalency among the attacking moieties [34]. This can be achieved with the radicals’ addition cycloaddition and halogenation of azomethine ylides. The cycloaddition of azomethine ylides (Prato reaction) is the most frequently used pathway. The surface modifications of defective carbon atoms are carried out through creating defects by oxidation to get -COOH moieties. These -COOH functionalities might be used to graft different specific groups including antibodies or protein and peptides via chemical reactions such as esterification or amidation. Several biological molecules [35], metals [36], and polymers [33] can be employed as the backbone of carboxylated CNTs. In covalent modifications of CNTs addition reactions were also adopted, which were carried out from those established for fullerenes or conventional for graphite surfaces [37, 38]. Besides of single group functionalization of CNTs, the plan of multi-functionalization (i.e. double or triple covalent modification) is probably significant, particularly for the delivery of drugs. The double covalent functionalization might be accomplished either through amidation/esterification and 1,3-dipolar cycloaddition; double arylation; double amidation/esterification; amidation and cyclopropanation; arylation and amidation; double 1,3-dipolar cycloaddition; 1,3-dipolar cycloaddition and arylation etc. Triple covalent functionalizations can be achieved through concurrent functionalizations with diverse aryl diazonium salts [39]. The above-discussed functionalizations, however, may be responsible for the loss of optical and electrical features, which are beneficial for recognizing CNTs in biological environments [18, 40]. In addition, a literature survey reported that the covalently surface functionalization associated with biological screening is a potential strategy to lower the CNTs’ toxicity [41].

14.3.2 Non-Covalent Functionalization Apart from covalent functionalization, noncovalent functionalization of CNTs permits preserving intrinsic optical and electrical properties.

CNTs for Treatment of Various Diseases  345 These modifications can be done through adsorption/wrapping of surfactants, polymers, and biopolymers on the tubular surface through π-π stacking and van der Waals interactions. Two strategies have been carried for non-covalent functionalizations of carbon nanotubes. The first one the CNT sidewall is wrapped by polymer chains [42] and the other is through π-π stacking interaction among π-electrons of graphite sheets and aromatic rings of the loaded materials on the surface of CNTs [43]. This non-­covalent functionalization will disrupt the van der Waals forces that ground SWNTs to be combined in the form of bundles [44] and may also enhance their water solubility. Non-covalent functionalizations of CNTs are also reported for the applications of medical. CNTs showed a good affinity for RNA and single-­stranded DNA and with the polynucleo­tide molecules develop an electrostatic complex, which may not only convey water miscibility but also be responsible for genes delivery within cells [11, 45]. A number of intrinsic physical characteristics of CNTs, such as Raman scattering and near-infrared fluorescence can be reserved and the conjugated electronic morphology of CNTs will not be hindered [46, 47]. Polymers such as poly (meta phenylene vinylene), tetra­alkylammonium, polyethylene glycol (PEG), poly (vinylpyrrolidone), and poly(oxyethylene), etc., when immersed in a solution containing polymer are able to wrap around the SWNTs [42]. The CNTs wrapped with polymer allow added support in the stabilization of CNTs dispersion. PEG is mainly employed for anchoring a broad variety of molecules (biomolecules, drugs, etc.). Due to its good solubility and biocompatibility under different physiological surroundings, PEG is widely employed for different causes in pharmaceutical applications [48]. PEGylated CNTs are proficient to reduce the uptake using the reticuloendothelial substrate, in mice block the binding of nonspecific serum proteins [49] and extend the blood circulating time [50]. In addition, poly(vinylpyrrolidone) used for anchoring drug molecules simplifies the process of bioimaging [18, 51]. The surfactants like Triton X-100, sodium dodecyl sulfate (SDS), and sodium dodecylbenzene sulfonate (SDBS), etc., adsorb onto the CNTs backbone using π-interactions to enhance their permeability, solubility, and dispersibility using biological membranes [52]. However advancement in the CNTs solubility can be lower (0.1 to 0.9 mg/mL), yet it is still suitable for biomedical application [52]. The CNTs solubilization in biomaterials (nucleic acids, proteins, and peptides) is a doubtlessly much appropriate technique to combine with biological substrates. It has been reported that a straightforward sonication with ­single-stranded DNA is the simplest route to solubilize CNTs. The complexes based on DNA-CNTs displayed good solubility and exceptional stability. Amphiphilic peptides (KV6-CONH2, A6RGD, RGD,

346  Functionalized CNT for Biomedical Applications EAK8, EAK12, and A6R etc.) exhibited a proficient approach to disperse CNT [53, 54]. M. Arnold et al. studied the noncovalent functionalization and dispersion of CNTs in water via peptide amphiphiles. The aqueous dispersibility of peptide amphiphiles-CNTs is because of the disclosure of hydrophilic heads to water whereas the hydrophobic tails are still attached to CNTs surface. The peptide amphiphiles-CNTs showed improved dispersibility in the aqueous phase along with offering bio-functionality to CNTs [55].

14.4 Toxicity/Bio-Safety Profile of Carbon Nanotubes The clinical trials of CNTs and their nanocomposites for biomedical applications are only possible when their security is fully confident and their toxicology in living systems is understood [56]. However, the literature survey based on toxicity reports on CNTs and biological behaviors toward biological systems suggested that the conflict in toxicity reports can be ascribed to various experimental variable conditions such as integrity of CNTs, cells, doses, and functionalizations of CNTs [57]. The toxicological behavior of CNT-based nanocomposites is mostly concentrated on different parameters such as impurities of metal, type and length of CNTs, and its functionalizations. The metal catalyst impurities originating during the growing process of CNTs are the main source of CNTs cytotoxicity [58]. In the current era of biomedicine, CNTs present a new promising platform whereupon various problems linked with traditional techniques of the therapeutic field are conveyed simultaneously [59]. Focusing on all the challenges researchers primarily harped about the goodness of CNTs based nanomaterials and insisted that no evident toxicity was noticed via CNTs [60]. Apart from this, few researchers affirmed that CNTs generated toxic behavior, mainly during pulmonary and intravenous incorporation of nanomaterials [61]. There was a discrepancy noticed in several reports, which has depressed its wide applications in the area of biomedical. It was absolutely apparent that a unique approach must be investigated for the fabrication technique of CNTs and the characterization requires to be carried out through valid approaches and models. Although the difficulties faced, the surface modification of CNTs using diethylentri amine pentaacetic dianhydride [23], functionalization with PEG [62], surface coating with vitamin E [63], manipulation with genetic materials [64], surface coating with immunoglobulins [65], conjugation with PEG and arginine-glycine-aspartate (RGD) peptides [23], and adhering to blood proteins [66] are the few techniques amid

CNTs for Treatment of Various Diseases  347

Table 14.1  Toxicity analyses of CNTs. S. no.

Carbon nanotubes

Dose

Exposure time

Model

Toxicity

Ref

1.

CNTs-OHPAA@ PEGMA

50 μL

12 or 24h

Cell lines HepG2

Cell viability can stay around 100% for 24 h. Displayed low good biocompatibility and cytotoxicity

[72]

2.

Pure CNTs

25 mg

4 weeks

Guinea pigs

CNTs does not promote any abnormalities of measurable inflammation or pulmonary function

[73]

3.

SWNT and MWNT

Large amount of CNT

24, 48, 72 and 96 h

Albino rabbits

No signs of health risk associated to allergic risk and skin irritation

[74]

4.

HiPco™ SWNT,

0.1 or 0.5 mg

Intratracheal instillation

7 or 90 days

Mortality was noticed with the over dose Stimulated dose-dependent epithelioid granulomas

[75]

5.

Pure laser SWNT

1 or 5 mg

Intratracheal instillation

From 24 h and up to 3 months

Within 24 h post-instillation the high dose produce mortality. Pulmonary inflammation with non dose dependent granulomas

[76]

(Continued)

348  Functionalized CNT for Biomedical Applications

Table 14.1  Toxicity analyses of CNTs. (Continued) S. no.

Carbon nanotubes

Dose

Exposure time

Model

Toxicity

Ref

6.

HiPco and SWNT

Aerosol

Human volunteers

30 min and 11–16 h

CNT in the range of 0.7 to 53 μg/m (HiPco material formed observable big clumps on the filter) Visible contamination (from 0.2 to 6 mg)

[77]

7.

SWNT

1.0. 40 and 200 mg

Mice

3 months

No noticeable hepatic harm is found in the histopathological analyses amid all the groups. There was no change in spleen

[78]

8.

Hat-stacked carbon nanofibers

-

Rats

1–4 weeks

Regular practice of inflammation for external bodies, with no strict inflammatory action. No inhibition of wound healing. No acute toxicity in the subcutaneous tissue.

[79]

9.

Functionalized -MWNTs

1 to 25 μg/mL

Liver cancer cells (HepG2)

24 h

Reduced the growth of cancer cell at a very low concentration.

[80]

™

3

CNTs for Treatment of Various Diseases  349 various others that showed achievement. To describe the CNTs in their purest form are particularly difficult to atomize in aqueous solutions and organic solvents. Hence, the toxicity of pure CNTs is more than modified CNTs, which hampers biological reactions. A suitable functionalization technique is required to prepare the CNTs dispersion in solvents through interacting with other molecules to the nanotubes’ surface [67]. In this respect, the covalent or noncovalent functionalization techniques can also minimize the toxicity associated with non-functionalized CNTs. Most of the toxicological reports are based on extraordinarily high doses and in vitro investigations with cell lines; therefore it is needed to be very careful to extrapolate these outcomes to living beings [68]. In recent times, advancement has been based on the elimination, biodistribution, and translocation of CNTs after precise introduction, producing the initial step for secure biomedical uses of CNTs [69]. It has been established [41, 70] that the biocompatibility and toxicity of CNTs can be monitored intentionally by material and chemical functionalizations [71]. A summary of a few previously reported studies describing the CNTs toxicity is displayed in Table 14.1.

14.5 Investigating the Promising Biomedical Effects of Functionalized CNTs CNTs exhibit exclusive properties which provide them for a wide range of applications in the field of biomedical science. Due to hydrophobic characteristics, CNTs are appropriate for several healthcare applications [81]. Although the capability to modify their surface (coatings, chemistry, and charge) and morphological (stiffness and aspect ratio of length and width) properties, the promising effects of CNTs are presently not completely understood [82]. Despite the definite application will signify the specific exposure to the human being [83], as previously reported, it is accepted that to humans the primary way of CNT exposure is through inhalation, and accordingly the lung is considered as the main organ of interest with respect to their consideration to endorse any unfavorable health issues or, alternatively, add to presented lung disease states [84]. The CNTs possess the ability to interact with the receptors of the cell surface which permits their internalization because of the occurrence of various functional sites. These receptor-mediated approaches permit specific cell loading that could assist the drop in the dose of a drug [85].

350  Functionalized CNT for Biomedical Applications

14.5.1 Functionalized CNTs-Based Remediation of Infectious Diseases Current breakouts of the rigorous acute respiratory syndrome (SARS), avian, and swine flu have shown that infectious diseases have emerged as serious global concerns related to the public health issue. Few infectious diseases, for instance, acquired immune deficiency syndrome (AIDS), have emerged as tedious, and no efficient remedies have been obtainable till now. The healthcare exercises of nanotechnology have been considered as a potential candidate in order to provide the fast diagnosis and efficient treatment of contagious diseases. Kang et al. [86, 87] reported that pure SWNTs attributed an antimicrobial potential in a size-dependent approach, suggesting that CNTs based materials can be assigned as potential aspirants for antimicrobial therapeutics. The surface modification of CNTs may create a position for the interaction of bioactive moieties, along with protecting the secondary structure [88] and, therefore, extract definite anti-epitope antibodies [89]. This type of coupling facilitated the recognition of antibodies to the conjugates. CNTs can play a vital role in the therapy of viral disease via producing high-sensitive detecting systems. As previously reported a coordinated biosensor [90] developed by SWNTs and gold nanoparticles and has been investigated for sensing the nanomolar range of human immunodeficiency virus-1 protease (HIV-1 PR), an aspartic protease accountable for maturation and assembly of virion [91]. The recognition of highly-delicate detection of this protease was encouraging to rapid fabrication of effectual HIV-1 PR inhibitors. On the other hand diagnosis of viral infection is the electrical sensing of hepatitis C virus RNA [92]. Distinctive electronic characteristics and a large surfaceto-­volume ratio produced CNTs a potential candidate for developing bio-detectors with the high-sensitivity, which were significantly required in the viral ailment diagnosis and the fabrication of fresh anti-viral drugs. Therefore, it is expected that CNTs can considerably offer to the nursing of infectious diseases in the future.

14.5.2 Functionalized CNTs for the Treatment of Central Nervous System Disorders (CNS) CNS disorders comprise brain tumors and neurodegenerative ailments. Due to the constrained anatomical contact (blood-brain barrier) and the complicated and unique environment of the CNS, it is further complicated to treat and diagnose CNS disorders in comparison to the other ailments. However, nanotechnology is the potential to modernize the rank quo in

CNTs for Treatment of Various Diseases  351 this area. Due to the nano dimensions and attainable exterior or external functionalizations, nanomaterials are capable to cross the blood-brain hurdle through different selected mechanisms and, therefore, they might work as valuable carriers for delivery towards selecting brain. Several types of nanomaterials have been successfully used as appropriate delivery devices for treating brain tumors and neurodegenerative defects [93, 94]. As a favorable therapeutic candidate, CNTs had been employed in neurosciences [95]. The outcomes of such investigations have revealed that both pure and chemically modified CNTs have a constructive influence on neuronal growth, while cytotoxicity was also demonstrated by them. It has been suggested that the charge on the CNT can be managed to regulate the outgrowth of neurite [96], and it has been investigated that functionalization of CNTs with brain derived neurotrophic factor or nerve growth aspect can stimulate neurons growth on the CNTs based scaffold [97]. Keefer et al. [98] confirmed that CNTs coated stainless steel and conventional tungsten wire electrodes can improve electrical stimulation as well as neurons recording in culture (monkeys and rats). The advantage could be assigned to the ability of CNTs to increase charge transfer and reduce the impedance of electrodes. Electrodes coated with CNT are suggested to enhance current and to support the construction of long-lasting brain-­ machine interfacing systems; which will assist the treatment and diagnosis of CNS disorders. The literature review also indicated that the fabrications of a few chemotherapeutic agents based on nanoparticles (e.g. doxorubicin (DOX) [99] possessed the probability for the systematized brain tumors’ chemotherapy with high efficiency than the reported agents. MWNTs functionalized with siRNA and DNA were addressed to be internalized through brain microglia (migratory monocytes resultant macrophage in the brain that is supposed to be an efficient site for the treatment of cancer) in vivo and in vitro without involving cytokine and proliferative modifications [100, 101]. These outcomes suggested that MWNTs can perform as a secure nano-carrier delivery source for immune therapies of brain cancers (e.g. gliomas). However, this research is still in its beginning stage, and further investigation is required.

14.5.3 Functionalized CNTs for Gene Delivery As a nanocarrier, the utilization of functionalized-CNTs for gene therapies is an emerging field of research interest that is presently under examination. In comparison to drugs based on small molecules, biomolecules including microRNA (miRNA), small interfering RNA (siRNA), and double stranded DNA (dsDNA), etc., are unable to go through the cellular membranes

352  Functionalized CNT for Biomedical Applications and simply deteriorate through nucleases [11, 102, 103]. However, various nonviral and viral vectors are present for the delivery of such nucleic acids, although controlling the immunogenicity of these biomolecules has grown as a major concern. In this regard, functionalized-CNTs look like capable delivery devices as functionalized-CNTs do not stimulate immune response inherently [89]. Recently it has also been found that CNTs can deliver bulky cargo loads of biomolecules to targeted positions. Under physiological conditions, the ammonium-functionalized CNTs are cationic in nature and also reported to translocate DNA and proficiently complex [104] within the cells [11, 105] and capable to deliver genes. Pantarotto et al. reported that ammonium-functionalized CNTs possess the ability to combine with plasmid DNA by electrostatic interactions [11]. The association with mammalian cells allows the ammonium-functionalized CNTs uptake into the cells through penetrating the cell membranes. The CNTs having reduced cytotoxicity and ammonium-functionalized CNTs coupled plasmid DNA is delivered to cells proficiently; the levels of gene expression are up to 10 times high than those accomplished with pristine DNA. This report permits consideration of this advanced technique as promising for gene delivery. It was also concluded that a unique permutation feature assignable to soluble CNTs evidence the probability of these structures as candidates of superior delivery systems for various therapeutic applications. The potential of CNTs for gene therapeutics has been further investigated for applications of gene silencing [106, 107]. Complexes of siRNA strands and SWNTs functionalized with a hydrocarbon tail were adopted for target killing of cancer cells. In addition advancements of this system are still essential to authenticate the in vivo use. Behnam et al. studied that functionalized SWNTs associated with polyethyleneimines accomplished enhanced transfection efficiency in comparison to underivatized polyethyleneimines [108]. A nearly 19-fold increase is being noticed in the functionalized-­SWNTs with the smallest polyethyleneimines. Additionally, polyethyleneimines-functionalized SWNTs were found competent gene delivery vectors in vivo along with tail vein injection in mice with the prevalent expression happening with the vector polyethyleneimines-functionalized-SWNTs using polyethyleneglycol as a linker. Pan et al. reported the fabrication of polyamidoamine dendrimer-­modified MWNTs for the delivery of genes into human cancer cells [109]. The polyamidoamine dendrimer modified MWNTs totally conjugated with fluorescein isothiocyanate (FITC)-labeled antisense c-myc oligonucleotides were incubated with liver cancer cell line liver hepatocellular carcinoma (HepG2) cells, breast cancer cell line (MDA-MB-435 cells), and MCF-7 cells (human breast), and revealed to penetrate into tumor cells within 15 min through laser

CNTs for Treatment of Various Diseases  353 confocal microscopy. This was observed that fabricated CNTs composites inhibited the dose-dependent means and cell growth in time followed by down-controlled the expression of the C-Myc protein and C-Myc gene. This report also favors that polyamidoamine dendrimer modified MWNTs (I)

A1

A2

B1

B2

60 54 48 41 35

(II)

Gut

Liver

Kidneys

Lungs

Spleen

Heart 600

A1

462 325 188

A2

50

B1

B2

Figure 14.1  (I) The fluorescently-labeled images for in vivo biodistribution (A) rCNTs and (B) fCNTs injected in mice. Images taken after the injection of 24 h of (A1) rCNTs, (A2) short rigid (s-rCNTs), (B1) fCNTs and (B2) short flexible (s-fCNTs). (II) The fluorescently labeled images for ex vivo organ biodistribution after 24 h of injection (A) rCNTs and (B) fCNTs. Fluorescent images overlaid with X-ray images regarding to the aggregation of (A1) rCNTs, (A2) s-rCNTs, (B1) fCNTs and (B2) s-fCNTs in the gut, liver, kidneys, lungs, spleen and heart. “Reprinted with permission from [110]. Copyright (2017) American Chemical Society.”

354  Functionalized CNT for Biomedical Applications composites have possibilities in applications for instance drug or gene delivery for molecular imaging and cancer treatment. Cifuentes-Rius et al. demonstrated rigid r-CNTs and flexible f-CNTs as promising delivery vectors because of their large aspect ratio and ability to translocate via plasma membranes as nanoneedle [110]. By paying close attention to the physicochemical characteristics of CNTs, r-CNTs and f-CNTs were coated using polyallylamine (ppAA) through plasma enhanced chemical vapor deposition. The ppAA-coated f-CNTs have been confirmed to bind larger amounts of plasmid-green fluorescent protein (pGFP) than r-CNTs, their tangled nature complicates the appropriate discharge of the loaded DNA and the internalization of the material into the cell. Fluorescence imaging of ex vivo and in vivo investigation can present a semi-quantitative determination of the CNTs response upon intravenous administration and permits observing of the similar matter at various time intervals. As shown in Figure 14.1 (I) after 24 hours of injection one mouse was imaged for each CNT group. The mice were culled and the main organs (lungs, gut, liver, kidneys, heart, and spleen) were promptly harvested and imaged, after the final time interval. Within the 24-h of this investigation, no side effects were evident in the mice injected with fluorescently labeled CNTs. Particularly, they suggested that that by selecting the r-CNTs over f-CNTs, accumulation might be eliminated, which is essential for the fruitful utilization of CNTs in gene and drug delivery [110].

14.5.4 Implication of Functionalized CNTs in Cancer Diagnosis and Treatment For cancer diagnosis and treatment, CNTs might be employed as a drug carrier as they possess the property of attaining the specific site followed by assembling in the cancer cells and forming the interaction with targeting moieties [111]. As a 1-dimensional structure CNTs might be employed for loading receptors that may associate through transducer and analyte as a result it can be used as biosensors which are more responsive to the chemical and electrical changes within the abrupt surrounding. As CNTs exhibit superb chemical stability, inertness, and mechanical strength [112] therefore they could be used as a reliable scaffold for tissue regeneration because it provides a gap to grow the cells and could be modified for stimulating orientation and growth of cells in an appropriate manner [113]. Shikori et al. prepared nanocomposite based on CNT and chitosan functionalized bioactive glass and employed for bone tissue regeneration with a sluggish biodegradability up to 30 days and compression strength of 5.95±0.5 MPa [114]. The fabricated nanocomposite scaffolds were tested on MG63

CNTs for Treatment of Various Diseases  355 osteoblast cell line, chitosan/bioactive glass/CNT nanocomposite in 80:20:2 wt% ratio found to be the finest ratio related to the cellular strength and viability. Therefore, describing that the horizons for using CNT nanocomposite scaffold could be improved by means of straightforward functionalization with definite biocompatible polymers [115]. The CNTs as a 3-dimensional network or in different arrangements can be employed for the immobilization of uniform protein. This can be defined through the investigation carried out via Song et al., where 3-dimensional CNT pillars were developed over silicon plate against which an oral squamous cell carcinoma and cytokeratin-19 antibody marker was immobilized. The developed system was sufficiently able to estimate cytokeratin-19 antibody even at nanogram level (0.5 ng/mL). Therefore noticeably suggests the significance of CNTs in constructing systems for detecting different biomarkers demonstrating the symptoms and traces of infection [116]. Moreover, CNTs are also being employed to improve the electric and thermoelectric transport features of several polymers, because of an important interaction among the polymer and CNTs [117]. It has been reported that CNT displays sensitivity regarding K-channels as per dose, this produces CNT as a feasible therapy for anticancer [118]. The CNTs applications are elaborated toward particular cancer therapy. Toward brain cancer, glioblastomas astrocytoma, and anaplastic are highly invasive and eminent, for such kind of tumor, polyethylene glycol along with SWNTs and fluorescein-labeled integrin monoclonal antibody has been employed [119]. An analysis carried out via Taghdisi et al. suggested that how functionalized-CNT can be used for targeting blood cancer [120]. This report suggested that when SWNTs loaded daunorubicin were functionalized through sgc8c aptamer an active targeting to the leukemic cells was feasible, therefore one more times signifying the limitless probabilities toward functionalization of CNT for disease targeting [120]. The CNTs in breast cancer have also been employed, which is found as responsible for cancer-causing death among women. Researchers employ the reality of over-expression of human epidermal growth factor receptor 2 (HER2)/ neureceptors, which has proliferated as targeted drug delivery. The modification of SWNT as a carrier toward small interfering (siRNA)-murine double minute 2 (MDM2) (siRNA-MDM2) and siRNA complexes was done for assessment in B-Cap-37 cells. Furthermore, the DOX loading in SWNTs coated with fluorescein and monoclonal antibody is utilized for colon cancer, which presented that in cancer cells it had specificity towards serum albumin receptors [121]. The primary cause of liver cancer includes hepatocellular carcinoma which is alcoholic cirrhosis, metabolic disorder, Hepatitis B, and Hepatitis C virus. CNTs conjugated dendrimer with

356  Functionalized CNT for Biomedical Applications antisense avian myelocytomatosis virus oncogene cellular homolog oligonucleotide displayed proficient drug delivery and targeting effect in the tumor cells [109]. This delivery of nucleic acids (anionic molecules) was facilitated owing to the functionalization of CNT with amine terminated dendrimers as they produced the necessary +ve charge which allowed proficient (~95%) entrapment of antisense avian virus oncogene. These studies describe the usefulness of surface functionalization of CNT which can utilize to obey the preferred effect [109]. Furthermore, MWNTs have also been employed in the treatment and diagnosis of cancer. Figure 14.2 displays the procedure of cancer treatment using MWNTs. In a study, dendrimer modified MWNTs were used for delivering doxorubicin drugs at lower pH [122]. It has also been reported that MWNTs are appropriate carriers for genes, peptides, and proteins as such macromolecules get simply deteriorated through enzymes found inside cells or on the cells surfaces. Guo et al. fabricated carrier cationic MWNTs-NH3+ employed to deliver the apoptotic siRNA against polo-like kinase (siPLK1) in calu6 tumor xenografts by direct intertumoral injections [123]. On the other side, it has been established by Wang et al. that narrow MWNTs of 9.2 nm (average diameter) had increased the affinity of non-reticular endothelial tissues, in comparison to broader MWNTs (average diameter 39.5 nm). This report also suggested that a larger aspect ratio of narrow MWNTs can be useful in the biomedical field because of the higher tissue accumulation [124].

Drug loading

MWNTs

Endocytosis

Drug Radiation

Degradation

Figure 14.2  Possible graphical illustration of cancer treatment through MWNTs.

CNTs for Treatment of Various Diseases  357

14.5.5 Functionalized CNTs for Drug Targeting and Release For designing a broad variety of drug delivery scaffolds CNTs have been employed for the treatment of several ailments. Definite exclusive properties of CNTs (adequate chemical stability, large surface area, high mechanical strength, and drug loading capacity) produce them brilliant nanocarriers for the delivery of different drugs [125]. Anticancer drug delivery scaffolds based on CNTs are frequent which implies specific targeting acquired by functionalization [126]. It can be calibrated sensitively to the microenvironment of the tumor which results in toxicity reduction along with reduce side effects. The prevention of cell proliferation and multi-drug resistance of tumor cells and the cell cycle can be possibly reduced in the affected cancer cells through the employment of a CNTbased scaffold [127]. The fabrication of peptide-functionalized SWNTs has revealed enhanced tumor-targeting and a high anti-tumor effect [85]. For immunization CNTs also behaves like a carrier against antigens [13]. Substances like acetylcholine are difficult to be transferred to the brain by means of normal procedure can be straightforwardly transported with the help of SWNT [128]. Although, covalent functionalization of SWNT with ester and amide bonds permits slow discharge of drug for a satisfactory period of the interval which sources the raise in the solubility of organic and aqueous phase [129]. MWNTs can be employed for association with neural tissue cells and are helpful for constructing competent drug and gene delivery sources [130]. The ability of SWNT towards the drugs’ inclusion is larger. The functionalized CNTs encapsulated with drug penetrate the cells via crossing the cell membrane through passive diffusion or by endocytosis and gets internalized within the nucleus and the cell organelles. Exocytosis and enzymatic degradation are the two main approaches that help CNTs to get rid of the cell [131]. The unique cocoon-like MWNTs and polyethylene glycol-based nanoparticles assign a large probability to be employed as nano-biomaterials and can be loaded simply with curcumin (a natural anticancer drug) [132]. Currently, in a recent report, the cisplatin (anticancer drug) has been investigated to have antileishmanial activity. The report suggested the enhanced antileishmanial activity and less cytotoxic nature of cisplatin bonded MWNTs at an especially lower amount highlighting the huge probability of CNTs as drug nanocarriers [133]. In another report, CNT buckypapers have been found to be constructive for transdermal drugs delivery. The in vitro analysis established the ability of CNT buckypapers in regulating the release of drugs [134]. Furthermore, CNTs can be covalently conjugated with small drug molecules for in vitro delivery. Drug cargoes and fluorescent dyes were concurrently associated

358  Functionalized CNT for Biomedical Applications to 1,3-dipolar cycloaddition modified CNTs through amide bonds for the anti-cancer drug delivery [135] or an antifungal drug [136] into cells. In an inspiring investigation, Moon et al. analyzed the in-vivo and in-vitro photothermal efficiency of polyethyleneglycol-conjugated SWNTs. The (a)

I PEG-SWNTs + NIR

II Untreated

III PBS + NIR

IV PEG-SWNTs

2 Day

14 Day

20 Day

I PEG-SWNTs + NIR

(b)

60 Day

3500 Mean Tumor Volume (mm3)

(c)

Untreated PEG-SWNTs PBS + NIR PEG-SWNTs + NIR

3000 2500 2000 1500 1000 500 0 0

5

10

15 Days

20

25

30

Figure 14.3  Photothermal effects of polyethyleneglycol (PEG)-SWNTs for destruction of tumor. (a) the mice treated I, PEG-SWNTs+NIR; II, untreated; III, PBS+NIR; IV, PEG+SWNTs. (b) Four mice treated with I, PEG-SWNTs+NIR after 60 days of treatments. (c) Time-dependent tumor growth curves of KB tumor cell xenografts. After sample treatments volumes of tumor were measured three times a week. “Reprinted with permission from [137]. Copyright (2009) American Chemical Society.”

CNTs for Treatment of Various Diseases  359

Table 14.2  Advanced and current drug/gene delivery systems based on CNTs. S. no.

CNTs material

Gene/drug

Application

Ref

1.

Polymer-doxorubicin-CNT

Doxorubicin

Targeted drug delivery for cancer

[139]

2.

Functionalized-CNT

Dacarbazine

Drug delivery system for the anticancer

[140]

3.

CNT-hydrogel

Rhodamine B and doxorubicin

Simultaneous monitoring and programmed release of dual drugs

[141]

4.

Functionalized-SWNT

Doxorubicin

Pathway to assemble a higher density of doxorubicin drug on the surfaces of functionalizedCNTs and its delivery

[142]

5.

Functionalized CNTs nanocarrier

Cladribine

Anti-cancer treatment

[143]

6.

Non-covalent functionalized SWNT-polyethyleneimines

Plasmid DNA

Competent gene delivery vectors in vivo as well as tail vein injection in mice

[108]

7.

Functionalized-MWNT

mRNA and C2C12 cells

The up-regulation in mRNA level in caspase 3/7 due to the presence of CNTs

[144]

(Continued)

360  Functionalized CNT for Biomedical Applications

Table 14.2  Advanced and current drug/gene delivery systems based on CNTs. (Continued) S. no.

CNTs material

Gene/drug

Application

Ref

8.

Functionalized-CNTs into hydrophobic drug crystals

Sulfamethoxazole and griseofulvin

Enhance aqueous solubility

[145]

9.

pH-responsive-functionalizedSWNT-polyethylenimine-betaine

doxorubicin and siRNA

Permutation of chemotherapy and gene therapy

[146]

10.

CNT-(Fe)/hydroxyapatite

Doxorubicin

Drug transport in high external magnetic fields (magnetic targeted delivery system)

[147]

11.

Chitosan-CNT based thermosensitive hydrogels

Methotrexate

Effectively controls growth of tumor cell

[148]

12.

Boron nitride/CNTs

Efavirenz

Anti-HIV drug delivery

[149]

13.

Functionalized SWNTs

Flutamide

Enhanced drug adsorption on the functionalized-CNT

[150]

14.

MWNT/lentinan/gemcitabine composite

Gemcitabine

Chemo-photothermal synergistic cancer treatment

[151]

15.

Oxidized MWNTs

Metformin

In vitro biological activity and inhibited cellular uptake of CNT

[152]

(Continued)

CNTs for Treatment of Various Diseases  361

Table 14.2  Advanced and current drug/gene delivery systems based on CNTs. (Continued) S. no.

CNTs material

Gene/drug

Application

Ref

16.

Cu/CNTs

Cu nanoparticles

Without any toxicity enhanced antimicrobial potential against biofilms

[153]

17.

Covalent-functionalized CNTs based drug carrier

Doxorubicin

Increased encapsulation efficiency

[154]

18.

Starch/MWNT/glucose

Zolpidem

For drug delivery

[155]

19.

Functionalized-MWNTs with nanoneedle structure

-

Gene delivery

[156]

20.

Functionalized-MWNTs/ cyclodextrins/polyethylenimine

Cidofovir

Antiviral drug delivery system

[157]

362  Functionalized CNT for Biomedical Applications fabricated system determined length in the range of 50–300 nm and diameter in the range of 2–5 nm. On the basis of the in vitro outcomes, further in vivo photo-thermal analysis was performed with nude mice bearing human epidermoid mouth carcinoma KB tumor cells. As demonstrated in Figure 14.3a, the mice medicated with polyethyleneglycol-conjugated SWNTs and near-infrared light (NIR) irradiation (I) represented fully demolition of tumors within twenty days. When treated with (I) a clear examination of black round marks was noticed on the mice skins (Figure 14.3a, I, 2 Day, and 14 Day). Figure 14.3 exhibits the destruction of tumor volume with control, polyethyleneglycol-SWNT+NIR, phosphate buffer saline (PBS)+NIR (PBS+NIR), and polyethyleneglycol-SWNT after 2, 14, and 20 days. Greater tumor destruction is shown in the case of polyethyleneglycol-SWNT+NIR in comparison to the untreated group [137]. From these investigations, it has been suggested that the functionalization of both SWNTs and MWNTs was effectively accomplished to deliver the payload to the targeting place. It was also noticed that CNTs in the functionalized form displayed enhanced antiproliferative effect and in-vitro drug release than the free drugs. The functionalization of polyethyleneglycol is also valuable to lower the toxicity problems that emerged due to the employment of CNTs [138]. Table 14.2 displayed the advanced and current applications of CNT as a drug/gene carrier, however, there are many further gene and drug delivery applications studied for CNTs.

14.6 Future Prospective CNTs open up an innovative era for different sensible purposes and are functional in fulfilling the fundamental physics at the non-metric level. CNTs acknowledged the various challenges on the way of development in the realistic application of these materials in the biomedical field. It also provides wonderful peculiar advantages to the next generation research by their progression in the chemicals, electronics, and approximately all sensible field of health care applications. In the present time, CNTs and their derivatives present genera of nanomaterials that are employed as biomaterials in the area of biomedicine and therapeutics. Furthermore, nanotechnology has been performed as a landmark in solving the restrictions and regulations. Therefore, size in nano-range along with the structural, electrical, and mechanical, properties of CNTs establish them as multifunctional nanocarriers, establishing CNTs functional candidates in research and developments of disease treatment, drug and gene delivery devices. There are various serious issues including cancer therapy targeted delivery

CNTs for Treatment of Various Diseases  363 of DOX [158], gene and drug delivery to the brain, that can be resolved with the help of CNTs. Confirmed targeted-tumor delivery with CNT is an approach for complex obstructions in cancer therapy. Literature survey revealed that CNT and its derivatives have been successfully used as targeted delivery scaffolds for drugs and gene delivery to the brain. Although further investigations is still needed to prepare functionalized CNT as a carrier device for proficient targeted brain therapy. Hence with the increasing demand, continuous researches are requisite to design multifunctional CNTs for effective and efficient drug delivery, for tissue engineering and stem cell therapy. However, functionalizations of CNTs reduce side effects, improve the bioavailability and physicochemical properties, but an absolute careful investigation of such biomaterials is still required. In addition, comprehensive understanding and safety knowledge of underlying physiological mechanisms of CNT incorporation is also essential. All above-­ reviewed strategies and approaches are only beneficial when the utilization CNTs will be sensibly effective, safe, along with obeying all safety and quality measures. There are more probabilities that drug delivery system based CNT and its derivatives will substitute most of the traditional imaging and diagnostic system. Despite the functionalized CNTs being valuable for deducting the cytotoxic effects and in offering improved bioavailability, it is essential to have a complete toxicological study of this nanocarrier. So far, among the present advanced drug delivery system based on CNTs, none of them were clinically approved. There is yet a further possibility to substitute all the traditional biomolecular detection and diagnostic systems through excellent and superspecific CNT-based systems in the future. Therefore, it can be suggested that in the future, with continuous research towards the advancement in CNTS, its utilization could reach heights in the field of biomedical.

14.7 Conclusion In summary, chemically functionalized CNTs with a tunable length and large surface area as well as exclusive physicochemical characteristics, make CNTs promising biomaterials in the field of biomedicine. The literature survey confirms that, the possibilities of CNTs as targeted drug delivery carriers have produced them capable materials for the medication and diagnosis of dangerous diseases such as CNS disorders, infectious diseases, and cancer. CNTs’ distinctive mechanical features also make them the main analytical topics in drug and gene targeting and release. Study on the possible toxicity of CNTs is still ongoing, but CNTs have been extensively

364  Functionalized CNT for Biomedical Applications established as a safer choice in comparison to the other nano-materials for example virus carriers and quantum dots. Thus, over the last two decades, the incredible growth of nanotechnology-based approaches in biomedical CNTs based materials has provided novel techniques for preparing unique functional materials. (i) Herein, a collection of reports have been summarized on different techniques and functionalized CNTs with the improved clinical investigation. (ii) With these approaches, desired molecules are combined with CNTs for specific selective imaging, drug delivery, and other therapies. (iii) As a new type of promising candidate, the biocompatibility towards bone, suitability for in vivo analyses, and toxicity of CNTs have been extensively reviewed on the basis of published reports. (iv) The literature survey revealed that, the composition of CNTs with particular targeting moiety or ligand that permits to improve the medication effect of the system is another fascinating research. (v) Additionally, the broadening of these practical techniques to the 2-dimensional forms of carbon specifically graphene is also nowadays a rapidly emerging area. Whereas the mission for exploring newly hybrid materials is constantly on and the study on CNT-based materials in distinct areas for instance doping is still open for discussion and investigations.

Important Websites https://now.northropgrumman.com/carbon-nanotube-applications-in-­ daily-life/ https://www.nanowerk.com/nanotechnology/introduction/introduction_ to_nanotechnology_22.php https://www.zdnet.com/article/5-surprising-uses-for-carbon-nanotubes/ https://en.wikipedia.org/wiki/Potential_applications_of_carbon_nanotubes

CNTs for Treatment of Various Diseases  365

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372  Functionalized CNT for Biomedical Applications 101. VanHandel, M., Alizadeh, D., Zhang, L., Kateb, B., Bronikowski, M., Manohara, H., Badie, B., Selective uptake of multi-walled carbon nanotubes by tumor macrophages in a murine glioma model. J. Neuroimmunol., 208, 3–9, 2009. 102. Karimi, M., Solati, N., Ghasemi, A., Hamblin, M.R., Carbon nanotubes part II: A remarkable carrier for drug and gene delivery. Expert Opin. Drug Deliv., 12, 1089–1105, 2015. 103. Kam, N.W.S. and Dai, H., Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc., 127, 6021–6026, 2005. 104. Lacerda, L., Pastorin, G., Wu, W., Prato, M., Bianco, A., Kostarelos, K., Luminescence of functionalized carbon nanotubes as a tool to monitor bundle formation and dissociation in water: The effect of plasmid-DNA complexation. Adv. Funct. Mater., 16, 1839–1846, 2006. 105. Singh, R., Pantarotto, D., McCarthy, D., Kostarelos, K., Binding and condensation of plasmid dna onto functionalized carbon nanotubes: Toward the construction of nanotube-based gene delivery vectors. J. Am. Chem. Soc., 127, 4388–4396, 2005. 106. Kam, N.W.S., Liu, Z., Dai, H., Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc., 127, 12492–12493, 2005. 107. Zhang, Z., Yang, X., Zhang, Y., Zeng, B., Wang, S., Zhu, T., Roden, R.B.S., Chen, Y., Yang, R., Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin. Cancer Res., 12, 4933–4939, 2006. 108. Behnam, B., Shier, W.T., Nia, A.H., Abnous, K., Ramezani, M., Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery. Int. J. Pharm., 454, 204–215, 2013. 109. Pan, B., Cui, D., Xu, P., Ozkan, C., Feng, G., Ozkan, M., Huang, T., Chu, B., Li, Q., He, R., Hu, G., Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems. Nanotechnology, 20, 125101, 2009. 110. Cifuentes-Rius, A., Boase, N.R.B., Font, I., Coronas, N., Ramos-Perez, V., Thurecht, K.J., Borrós, S., In vivo fate of carbon nanotubes with different physicochemical properties for gene delivery applications. ACS Appl. Mater. Interfaces, 9, 11461–11471, 2017. 111. Ali, Z., Abul-faraj, A., Li, L., Ghosh, N., Piatek, M., Mahfouz, M.M., Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol. Plant, 8, 1288–1291, 2015. 112. Geng, Y., Liu, M.Y., Li, J., Shi, X.M., Kim, J.K., Effects of surfactant treatment on mechanical and electrical properties of CNT/epoxy nanocomposites. Compos. Part A Appl. Sci. Manuf., 39, 1876–1883, 2008. 113. Kharaziha, M., Shin, S.R., Nikkhah, M., Topkaya, S.N., Masoumi, N., Annabi, N., Dokmeci, M.R., Khademhosseini, A., Tough and flexible CNT–polymeric

CNTs for Treatment of Various Diseases  373 hybrid scaffolds for engineering cardiac constructs. Biomaterials, 35, 7346– 7354, 2014. 114. Shokri, S., Movahedi, B., Rafieinia, M., Salehi, H., A new approach to fabrication of Cs/BG/CNT nanocomposite scaffold towards bone tissue engineering and evaluation of its properties. Appl. Surf. Sci., 357, 1758–1764, 2015. 115. Chandrasekhar, P., Conducting polymers, fundamentals and applications, pp. 61–64, Springer International Publishing, Cham, 2018. 116. Liu, P., Modifications of carbon nanotubes with polymers. Eur. Polym. J., 41, 2693–2703, 2005. 117. Wang, Q., Yao, Q., Chang, J., Chen, L., Enhanced thermoelectric properties of CNT/PANI composite nanofibers by highly orienting the arrangement of polymer chains. J. Mater. Chem., 22, 17612, 2012. 118. Ortega-Guerrero, A., Espinosa-Duran, J.M., Velasco-Medina, J., TRPV1 channel as a target for cancer therapy using CNT-based drug delivery systems. Eur. Biophys. J., 45, 423–433, 2016. 119. Zhou, F., Xing, D., Ou, Z., Wu, B., Resasco, D.E., Chen, W.R., Cancer photothermal therapy in the near-infrared region by using single-walled carbon nanotubes. J. Biomed. Opt., 14, 021009, 2009. 120. Taghdisi, S.M., Lavaee, P., Ramezani, M., Abnous, K., Reversible Targeting and controlled release delivery of daunorubicin to cancer cells by ­aptamer-wrapped carbon nanotubes. Eur. J. Pharm. Biopharm., 77, 200–206, 2011. 121. Cortazar, P., Zhang, L., Untch, M., Mehta, K., Costantino, J.P., von Minckwitz, G., Pathological complete response and long-term clinical benefit in breast cancer: The CTNeoBC pooled analysis. Lancet, 384, 164–172, 2014. 122. Wen, S., Liu, H., Cai, H., Shen, M., Shi, X., Targeted and pH-responsive delivery of doxorubicin to cancer cells using multifunctional dendrimer-modified multi-walled carbon nanotubes. Adv. Healthc. Mater., 2, 1267–1276, 2013. 123. Guo, C., Al-Jamal, W.T., Toma, F.M., Bianco, A., Prato, M., Al-Jamal, K.T., Kostarelos, K., Design of cationic multiwalled carbon nanotubes as efficient siRNA vectors for lung cancer xenograft eradication. Bioconjug. Chem., 26, 1370–1379, 2015. 124. Wang, J.T.-W., Fabbro, C., Venturelli, E., Al-Jamal, K.T., The relationship between the diameter of chemically-functionalized multi-walled carbon nanotubes and their organ biodistribution profiles in vivo. Biomaterials, 35, 9517–9528, 2014. 125. Chou, C.-C., Hsiao, H.-Y., Hong, Q.-S., Chen, C.-H., Peng, Y.-W., Chen, H.-W., Yang, P.-C., Single-walled carbon nanotubes can induce pulmonary injury in mouse model. Nano Lett., 8, 437–445, 2008. 126. Huang, Y., Liang, J., Chen, Y., The application of graphene based materials for actuators. J. Mater. Chem., 22, 3671–3679, 2012. 127. Cheng, J., Meziani, M.J., Sun, Y.-P., Cheng, S.H., Poly(ethylene glycol)-­ conjugated multi-walled carbon nanotubes as an efficient drug carrier for

374  Functionalized CNT for Biomedical Applications overcoming multidrug resistance. Toxicol. Appl. Pharmacol., 250, 184–193, 2011. 128. Yang, Z., Zhang, Y., Yang, Y., Sun, L., Han, D., Li, H., Wang, C., Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomed. Nanotechnol. Biol. Med., 6, 427–441, 2010. 129. Khazaei, A., Organic functionalization of single-walled carbon nanotubes (SWCNTs) with some chemotherapeutic agents as a potential method for drug delivery. Int. J. Nanomed., 5, 639, 2010. 130. Bardi, G., Nunes, A., Gherardini, L., Bates, K., Al-Jamal, K.T., Gaillard, C., Prato, M., Bianco, A., Pizzorusso, T., Kostarelos, K., Functionalized carbon nanotubes in the brain: Cellular internalization and neuroinflammatory responses. PLoS One, 8, 80964, 2013. 131. Costa, P.M., Bourgognon, M., Wang, J.T.-W., Al-Jamal, K.T., Functionalised carbon nanotubes: From intracellular uptake and cell-related toxicity to systemic brain delivery. J. Control. Release, 241, 200–219, 2016. 132. R., H., M., J., Haridoss, P., Sharma, C.P., Novel nano-cocoon like structures of polyethylene glycol–multiwalled carbon nanotubes for biomedical applications. Nano-Struct. Nano-Objects, 13, 30–35, 2018. 133. Akhtari, J., Faridnia, R., Kalani, H., Bastani, R., Fakhar, M., Rezvan, H., Beydokhti, A.K., Potent in vitro antileishmanial activity of a nanoformulation of cisplatin with carbon nanotubes against Leishmania major. J. Glob. Antimicrob. Resist., 16, 11–16, 2019. 134. Schwengber, A., Prado, H.J., Zilli, D.A., Bonelli, P.R., Cukierman, A.L., Carbon nanotubes buckypapers for potential transdermal drug delivery. Mater. Sci. Eng. C, 57, 7–13, 2015. 135. Pastorin, G., Wu, W., Wieckowski, S., Briand, J.-P., Kostarelos, K., Prato, M., Bianco, A., Double functionalisation of carbon nanotubes for multimodal drug delivery. Chem. Commun., 11, 1182, 2006. 136. Wu, W., Wieckowski, S., Pastorin, G., Benincasa, M., Klumpp, C., Briand, J.-P., Gennaro, R., Prato, M., Bianco, A., Targeted delivery of amphotericin B to cells by using functionalized carbon nanotubes. Angew. Chem., 117, 6516–6520, 2005. 137. Moon, H.K., Lee, S.H., Choi, H.C., In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes. ACS Nano, 3, 3707– 3713, 2009. 138. Jafari, M., Heidari, D., Ebrahimnejad, P., Synthesizing and characterizing functionalized short multiwall carbon nanotubes with folate, magnetite and polyethylene glycol as multitargeted nanocarrier of anti-cancer drugs. Iran. J. Pharm. Res., 15, 449–456, 2016. 139. Kamath, A., Laha, A., Pandiyan, S., Aswath, S., Vatti, A.K., Dey, P., Atomistic investigations of polymer-doxorubicin-CNT compatibility for targeted cancer treatment: A molecular dynamics study. J. Mol. Liq., 348, 118005, 2022.

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15 Role of Functionalized Carbon Nanotubes in Antimicrobial Activity: A Review Monika Aggarwal1, Samina Husain2* and Basant Kumar1 Department of Physics, Maharaja Agrasen Institute of Technology (affiliated to GGSIPU), New Delhi, India 2 Centre for Nanoscience and Nanotechnology, Jamia Millia Islamia, New Delhi, India 1

Abstract

CNTs one of the novel carbon materials with its unique structure has certainly drawn attention of the researchers across the globe. Currently, much of the research enthusiasm has been shown for drug delivery, gene delivery, vaccine delivery and immense biomedical applications are due to the high surface area of CNTs that is capable of absorbing or conjugating with several therapeutic molecules. However, toxicity of CNTs in both human and animal cells, their hydrophobic nature, biocompatibility, solubility and dispersibility of CNTs in both organic and aqueous vehicles is a major concern. The lateral parameters especially cause agglomeration or clustering thereby altering the properties of CNTs and hence so forth this can be overcome with the use of functionalized Carbon Nanotubes (f-CNTs). An insight to CNT surface chemistry modification, as the surfaces coated with CNTs depicts antimicrobial activities, would be taken up in the chapter. The chapter will focus on introduction and overview of f-CNTs in which specific conjugation of bioactive molecules takes place with a variety of functional or chemical groups thereby enhancing the specific targeting. The chapter will focus on the possible mechanisms of both functionalized SWNTs and MWNTs antimicrobial activities so that they can be explicitly used in various biomedical applications. Keywords:  Antimicrobial activity, functionalized carbon nanotubes, single-wall CNTs, multi-wall CNTs

*Corresponding author: [email protected] Jeenat Aslam, Chaudhery Mustansar Hussain and Ruby Aslam (eds.) Functionalized Carbon Nanotubes for Biomedical Applications, (377–412) © 2023 Scrivener Publishing LLC

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15.1 Introduction Life exists in the universe only because the carbon atom possesses certain exceptional properties (James Jeans)

Carbon, derived from the Latin word “carbo”, has been acknowledged for a variety of applications and is the key ingredient for 95 percent of known compounds. It is the basic building block with a remarkable and unique property of showing strong affinity for bonding with itself and with different atoms. Materials research and technology have paved the way for exploring the relation between the structure and properties of a variety of diverse wellknown materials as well as processes for altering the structure and properties by taking care of the total control over the parameters. As a consequence, we have come across many unique and fascinating results not only for carbon but for its allotropes too. The bulk carbon materials like diamond and graphite have been known for decades but introduction of quasi-low-­ dimensional allotropes of carbon like fullerenes, carbon nanotubes and the two-­dimensional graphene [1] completely gathered attention worldwide and set new area for explicit interdisciplinary research in physics, chemistry, material sciences, biomedical sciences etc. The unparalleled properties of these dynamic materials over conventional materials have really urged the desire for more and more research from researchers and scientists across the globe.

15.2 Introduction to CNTs Carbon nanotubes (CNTs) are one of the greatest discoveries by Ijima in 1991 [2]. An allotrope of graphite, it is highly stable and a promising material for a variety of technological applications and so a major reason for extensive research in this area. As compared to the parent graphite material, CNTs exhibit a range of many unexpected phenomenon thereby increasing the expectation of the researchers to further explore the materials to the fullest. Their outstanding properties viz. small size, imperfection in their structures, metallic or semi-conducting property, their high thermal conductivity of about 1900 Wm-1K-1 when compared to diamond is twice [3, 4], thermal stability is up to 2800°C and electrical conductivity is about 103 S cm-1 with 1000 times higher electric current capacity, high elastic modulus (>1 TPa), high mechanical strength than other materials (around 10–100 times greater than the strongest steel) [5] have captured interest worldwide.

Role of CNTs in Antimicrobial Activity  379 Documents by year 14

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Figure 15.1  Last 5-year development in the field of CNTs/f-CNTs anti-microbial activity.

Figure (15.1) shows the last five-year development in the field of CNTs/fCNTs-anti-microbial activity. The graph depicts the research publications on CNTs/f-CNTs for antimicrobial activity documented during last five years (2018–2022).

15.2.1 Classification of CNTs Nanotubes can be broadly classified as: Carbon Nanotubes (CNTs)

Single-Walled Carbon Nanotubes (SWCNTs)

Multi-Walled Carbon Nanotubes (MWCNTs)

a) Single-wall carbon nanotubes (SWCNTs) Its structure is like a seamless cylinder formed by wrapping up of one-atom thick layer of graphite which is called graphene (Figure 15.2(a)). With tube length being thousands of times larger and diameters ranging from 0.4 to 2–3 nm, SWNTs can be up to the order of cms. They are arranged hexagonally in bundles (Figure 15.2(b)) to form crystal-like structure [6] which exhibits unique electrical properties not shared by MWNT variants. These are less expensive to be produced and are good conductors, thus forming the basic part of electronics.

380  Functionalized CNT for Biomedical Applications

(a) A SWCNT

(b) Bundles of SWCNTs

Figure 15.2  (a) A SWCNT (b) Bundles of SWCNTs. Reproduced with permission from [17].

b) Multi-walled carbon nanotubes (MWCNTs) Multi-walled nanotubes (MWCNTs) are concentric layer of graphitic sheets rolled into a tube [7]. These are rolled around one common axis with a hollow centre (Figure 15.3(a, b)) with the interlayer spacing of ~0.34 nm close to that of graphite. The inner and outer diameter can vary from 0.4 nm up to a few nm and from 2 nm up to 20–30 nm respectively according to the number of layers. Due to the

Small-sized MWCNTs Long MWCNTs

(a) MWCNT structure

(b) MWCNTs, as observed by HRTEM

Figure 15.3  (a) MWCNT structure. (b) MWCNTs, as observed by HRTEM. Reproduced with permission from [2].

Role of CNTs in Antimicrobial Activity  381 presence of pentagonal defects, nanotubes exhibit closed tips with their axial size ranging from 1μm to a few cm. The structure of MWCNTs can be interpreted in terms of two models: the Russian Doll model wherein there are concentric sheets of graphite cylinders and the Parchment model wherein a single layer of graphite is rolled around itself to form a tube [8–10].

15.2.2 Structure of CNTs Structural classification of these novel materials is an interesting challenge. Being crystalline in nature, many fundamentals and theories cannot be applied to these tubular structures using the conventional crystallography for three-dimensional solids. Carbon nanotube (CNT) is tube of a few nanometers in diameter which is many microns long and made up of rolling of a sheet of hexagonal ­honeycomb carbon lattice (Figure 15.4). Few of its unique features include: • Infinite length • Presence of all the atoms on the surface • Resemblance to graphene (single graphitic layer) Though the binding is same in CNTs and graphite but CNTs are made up of rolling up of one or more sheets of graphene into a tubular structure. From the perpendicular view of the graphitic layers, we can observe ­honeycomb pattern of graphite. Turning back these patterns on top of themselves, edges can be joined keeping one end closed while the other to form a tube of graphite namely called as a nanotube (Figure 15.5). CNTs popularly known as “wondrous material” possess unique, remarkable and extraordinary properties. The world-wide large-scale production of CNTs is due to the simplest chemical configuration with atomic covalent bonding and its ever-ending demand has fascinated us in all respect. CNTs can be synthesized on large scale with a variety of techniques. The oldest

Figure 15.4  Structure of a CNT. Reproduced with permission from [17].

382  Functionalized CNT for Biomedical Applications

Hexagonal structure of a carbon atom

Graphene sheet

Rolling of a graphene sheet

Formation of a CNT

Figure 15.5  Schematic of formation of a CNT.

method known so far is the Arc-Discharge that was employed initially for the synthesis of carbon filaments and fibers. This method was used in the early 60s. R. Bacon used this technique for the synthesis of carbon fibers called whiskers, in nineties by Krätschmer and Huffman for the large-scale production of fullerenes, and later after improvisation, the technique was useful for the synthesis of MWCNTs and SWCNTs. Other techniques like Laser Ablation or Chemical Vapour Deposition (CVD) also marked their importance and relevance in producing CNTs. Economically Chemical Vapor Deposition (CVD) has proved to be one of the best methods because of its unique attributes: simple to operate, controlled process, energy efficient, simple raw materials used, high scalability, high yield and purity. Low temperature (